AP-1 activation through endogenous H(2)O(2) generation by alveolar macrophages

Reactive oxygen species released during the respiratory burst are known to participate in cell signaling. Here we demonstrate that hydrogen peroxide produced by the respiratory burst activates AP-1 binding. Stimulation of the macrophage cell line NR8383 with respiratory burst agonists ADP and C5a increased AP-1 binding activity. Importantly, this increase in binding was blocked by catalase, confirming mediation by endogenous H₂O₂. Moreover, exogenously added H₂O₂ mimicked the agonists, and also activated AP-1. Antibodies revealed that the activated AP-1 complex is composed predominantly of c-Fos/c-Jun heterodimers. Treatment of the cells with ADP, C5a and H₂O₂ (100 μM) all increased the phosphorylation of c-Jun. c-Fos protein was increased in cells treated with C5a or high dose (200 μM) H₂O₂, but not in cells treated with ADP. The MEK inhibitor, PD98059, partially blocked the C5a-mediated increase in AP-1 binding. A novel membrane-permeable peptide inhibitor of JNK, JNKi, also inhibited AP-1 activation. Together these data suggest that C5a-mediated AP-1 activation requires both the activation of the ERK and JNK pathways, whereas activation of the JNK pathway is sufficient to increase AP-1 binding with ADP. Thus, AP-1 activation joins the list of pathways for which the respiratory burst signals downstream events in the macrophage.     

Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

Plasma membrane potential interferes with the respiratory burst of peripheral granulocytes

Membrane potential is involved in the regulation of several immune functions developed by granulocytes. The Na⁺/K⁺ gradient across the plasma membrane, mainly generated by the Na⁺/K⁺ pump, plays a key role in the maintenance of membrane potential. This study is focused on the correlation between plasma membrane potential and the in vitro receptor - triggered respiratory burst of normal human peripheral granulocytes. The respiratory burst was measured as superoxide anion release by the cytochrome c reduction test and plasma membrane potential was modulated by experimental changes of the extracellular potassium concentration. Results show a differentiated cellular response, depending on the in vivo activation state and on the signals received in vitro by granulocytes via CR3 or FcγR. Alteration of the membrane potassium gradient modulates the respiratory burst of unstimulated and CR3-activated cells, whilst it does not seem to significantly interfere with the signals delivered by FcγR.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Serotonin Acts as a Radical Scavenger and Is Oxidized to a Dimer During the Respiratory Burst of Activated Microglia

Abstract: Serotonin (5-HT) is known to be readily oxidized and to act as a scavenger of reactive oxygen species produced, e.g., in the presence of peroxidase and H₂O₂ or during the respiratory burst of phagocytes. One major oxidation product formed under these conditions, the 5-HT dimer 5,5'-dihydroxy-4,4'-bitryptamine (DHBT), was suggested to have neurotoxic properties and to contribute to neuronal damage in neurodegenerative disorders. It is shown in the present study that the luminol-enhanced chemiluminescence signal measured after stimulation of the respiratory burst activity of cultivated rat microglial cells by the addition of phorbol 12-myristate 13-acetate is suppressed by 5-HT in a dose-dependent manner. During this process, 5-HT is oxidized to DHBT. Neither the intraventricular injection of DHBT nor the addition of DHBT to cultured astrocytes, neurons, or PC-12 cells was found to cause measurable cytotoxic effects. It is concluded that extracellular 5-HT locally released from platelets and 5-HT nerve endings at sites of brain damage or inflammation, through its suppressant effect on the release of reactive oxygen species during the respiratory burst of activated microglia, may contribute to attenuate secondary tissue damage in the CNS.     

Erythrocyte membranes inhibit respiratory burst and protein nitration during phagocytosis by macrophages

Phagocytosis is associated with respiratory burst producing reactive oxygen and nitrogen species. Several studies imply that erythrocytes can inhibit the respiratory burst during erythrophagocytosis. In this work we studied the mechanisms of this effect using control and in vitro peroxidized erythrocyte membranes. We demonstrated that autofluorescence of peroxidation products can be used for visualization of phagocytozed membranes by fluorescence microscopy. We also found that respiratory burst induced by a phorbol ester was inhibited by control membranes (5 mg/ml) to 63 % (P < 0.001), and to 40 % by peroxidized membranes (P < 0.001). We proved that this effect is not caused by the direct interaction of membranes with free radicals or by the interference with luminol chemiluminescence used for the detection of respiratory burst. There are indications of the inhibitory effects of iron ions and free radical products. Macrophages containing ingested erythrocyte membranes do not contain protein-bound nitrotyrosine. These observations imply a specific mechanism of erythrocyte phagocytosis.     

灵芝子实体中不同极性的三萜体外抗肿瘤及抗炎活性比较

为了解灵芝中不同极性三萜活性的差异,运用大孔树脂将灵芝总三萜根据极性大小进行分段,采用高效液相法分析各极性部分的HPLC指纹图谱和三萜含量,并比较其对不同肿瘤细胞增殖的抑制作用和抗炎活性。结果显示D-101大孔树脂可以有效地将灵芝总三萜分为中等极性和低极性两部分,两部分的三萜含量分别为(279.00±2.90)mg/g、(94.52±2.03)mg/g。其中,低极性三萜的体外抗肿瘤活性明显强于中等极性三萜,其对K562、SW620、L1210细胞增殖抑制的IC₅₀值分别为(74.12±1.94)μg/mL、(121.45±2.13)μg/mL、(13.52±1.13)μg/mL。另外,低极性三萜对RAW264.7细胞呼吸爆发的抑制作用也明显强于中等极性三萜。进一步对低极性三萜和中等极性三萜单体的抗炎活性进行比较,发现低极性三萜灵芝烯酸F、灵芝酸DM、灵芝醇B对RAW264.7细胞呼吸爆发的抑制作用明显强于极性较高的灵芝酸C₂、灵芝酸A。该研究阐明了灵芝子实体中不同极性三萜部位的抗肿瘤及抗炎活性并不相同,为将来新药开发和灵芝质量标准的改进建立了基础。     

Influence of copper-(II) on colloidal carbon-induced kupffer cell-dependent oxygen uptake in rat liver: Relation to hepatotoxicity

Formation of reactive O₂ species in biological systems can be accomplished by copper-(II) (Cu²⁺) catalysis, with the consequent cytotoxic response. We have evaluated the influence of Cu²⁺ on the respiratory activity of Kupffer cells in the perfused liver after colloidal carbon infusion. Studies were carried out in untreated rats and in animals pretreated with the Kupffer cell inactivator gadolinium chloride (GdCl₃) or with the metallothionein (MT) inducing agent zinc sulphate, and results were correlated with changes in liver sinusoidal efflux of lactate dehydrogenase (LDH) as an index of hepatotoxicity. In the concentration range of 0.1-1 μM, Cu²⁺ did not modify carbon phagocytosis by Kupffer cells, whereas the carbon-induced liver O₂ uptake showed a sigmoidal-type kinetics with a half-maximal concentration of 0.23 μM. Carbon-induced O₂ uptake occurred concomitantly with an increased LDH efflux, effects that were significantly correlated and abolished by GdCl₃ pretreatment or by MT induction. It is hypothesized that Cu²⁺ increases Kupffer cell-dependent O₂ utilization by promotion of the free radical processes related to the respiratory burst of activated liver macrophages, which may contribute to the concomitant development of hepatocellular injury.     

Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

Pancreatic phospholipase A2--mediated enhancement of the respiratory burst response of human neutrophils

The aim of this study was to investigate the effects of exogenously added pancreatic phospholipase A2 (pPLA2) on the production of reactive oxygen species by human polymorphonuclear leukocytes (PMNs). Pancreatic PLA2 was used because PMNs do not possess a receptor for that enzyme and, therefore, the receptor-mediated effects could be excluded. Respiratory burst activity of PMNs was monitored by luminol-amplified chemiluminescence and the lipid composition of neutrophils after treatment with pPLA2 was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that the products of the pPLA2 digestion of the PMN membrane--lysophospholipids and the corresponding free fatty acids--significantly enhanced the respiratory burst response of human neutrophils.     

Superoxide anion burst and taxol production induced by Ce in suspension cultures of Taxus cuspidata

Generation of reactive oxygen species (ROS) induced by Ce⁴⁺ in suspension cultures of Taxus cuspidata was investigated. The burst of superoxide anions (O₂√⁻) occurred rapidly after the addition of Ce⁴⁺ and reached maximum at 4.3 h, while the total level of the cellular reactive oxygen species maintained unchanged. The intracellular superoxide dismutase (SOD) and catalase (CAT) were activated while the intra/extracellular peroxidases (PODs) were inhibited accompanying the O₂√⁻ burst. The pretreatment of the suspension cultures with diphenylene iodonium (DPI), a suicide inhibitor of the NADPH oxidase, blocked the O₂√⁻ burst, inhibiting the cell apoptosis and taxol production induced by Ce⁴⁺. These results show that NADPH oxidase played a key role in O₂√⁻ burst and O₂√⁻ served as a mediator of Ce⁴⁺ for cell apoptosis and taxol production. The pretreatments of the suspension cultures with anthracene-9-carboxylate, an ion-channel blocker, nifedipine, a Ca²⁺-channel blocker, neomycin, a phospholipase C (PLC) inhibitor, or suramin, a G-protein inhibitor, decreased O₂√⁻ burst induced by Ce⁴⁺. It is thus inferred that Ce⁴⁺-induced O₂√⁻ burst, which mediated cell apoptosis and taxol production by activating the ion-channels, PLC, G-proteins and NADPH oxidase.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

Turning on the respiratory burst

The respiratory burst is a distinguishing property of phagocytes. It is induced by chemotactic stimulation or phagocytosis and reflects the activation of a membrane-bound enzyme system that transfers electrons from cytosolic NADPH to extracellular oxygen, producing superoxide. The products of the burst are essential for the killing of microorganisms, but are also a cause of tissue damage and inflammation. Studies aimed at a better understanding of the regulation of the respiratory burst should help in the search for new ways to treat infections and inflammation.     

Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils: Effects of recombinant human colony stimulating factors and dihydrocytochalasin B

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O₂ ⁻ production by neutrophils. We confirmed a time dependent augmentation of O₂ ⁻ production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O₂ ⁻ production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O₂⁻-production that was first evident at 2 hours. The fall in O₂⁻-forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O₂⁻, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.     

Stimulation of the respiratory burst in peripheral blood monocytes by lipoteichoic acid. The involvement of calcium ions and phospholipase A2

Lipoteichoic acid (LTA) from Streptococcus faecalis stimulates the respiratory burst in peripheral blood monocytes (mon), as measured by cytochrome C reduction. The effect of LTA was time and dose dependent. LTA stimulated the respiratory burst in a biphasic manner within a range of 1 to 1000 ng/ml.10(6) mon, with maximal activity at 50 ng/ml. At this concentration LTA increased the activity from 0.97 +/- 0.2 to 4.88 +/- 0.2 nmol.10(6) mon/20 min. The role of calcium ions in the effect of LTA in stimulating respiratory burst was studied by changing the availability of calcium ions in the medium, and by measuring the effect of LTA on 45Ca2+ uptake and on intracellular Ca2+ levels. Removal of extracellular calcium ions in the presence of the calcium chelator EGTA, abolished the LTA-stimulated respiratory burst. LTA (50 ng/ml) was found to increase 45Ca2+ uptake into monocytes within seconds (from 2200 +/- 242 in the untreated cells to 4642 +/- 365 cpm/min in the LTA-treated mon). At this concentration, LTA stimulated an immediate rise in the intracellular free Ca2+ concentration to 155 +/- 15 nM as compared with 120 +/- 14 nM in the unstimulated monocytes. LTA caused a specific release of arachidonic acid indicating the involvement of phospholipase A2 in the transduction signal stimulating the respiratory burst by LTA.     

Antioxidative activity of flavonoids in stimulated human neutrophils

The release and production of oxidative products generated by the respiratory burst under the influence of natural flavonoids: quercetin, kaempferol and isorhamnetin derivatives have been studied in the polymorphonuclear neutrophils (PMNs) from healthy human donors. Flavonoids were tested in vitro at concentration range 1-100 microM. The antioxidative potential of flavonoids was compared to the activity of a food preservative, butylated hydroxyanisole. Two methods were applied to the measurement of the PMNs respiratory burst: flow cytometry using dichlorofluorescein diacetate and luminol-dependent chemiluminescence. It was found that the studied products decreased the neutrophil hydrogen peroxide production in concentration-dependent manner. The highest degree of inhibition was registered for concentration of 100 microM, although in the chemiluminescence method the metabolic activity inhibition was more prominent. Antioxidative activity of flavonoids depended on the number of hydroxyl groups. These results provide useful data for establishing methods used to assess the respiratory burst in phagocytic cells.     

Zymosan-induced luminol-amplified chemiluminescence of whole blood phagocytes in experimental and human hyperthyroidism

Luminol-amplified CL of whole blood phagocytes was studied in rats given 3 consecutive doses of 0.1 mg L-triiodothyronine T₃/kg or in hyperthyroid patients, after stimulation by zymosan. In both cases, CL was significantly increased, in effect which was produced independently of the opsonization of the zymosan particles and markedly inhibited by azide. The in vitro addition of T₃ or L-thyroxine (T₄) to whole blood phagocytes from normal rats did not modify the opsonized zymosan-dependent CL, when assayed at the concentrations found in eutrhyroid subjects or in hyperthyroid patients. Administrations of propylthiouracil (400 mg/day for 2–3 months) to hyperthyroid patients reduced the CL response observed prior to treatment, to values comparable to those found in the euthyroid group. These data indicate that hyperthyroidism elicits an enhanced respiratory burst activity of whole blood phagocytes, probably related to adaptive changes induced by thyroid hormone on the mieloperoxidase-H₂O₂ system, rather than to direct actions of the hormone molecule or changes in the opsonic capacity of plasma.     

Simulated microgravity impairs respiratory burst activity in human promyelocytic cells

Summary The concept of microgravity (free-fall) influencing cellular functions in nonadherent cells has not been a part of mainstream scientific thought. Utilizing rotating wall vessels (RWVs) to generate simulated microgravity conditions, we found that respiratory burst activity was significantly altered in nonadherent promyelocytic (HL-60) cells. Specifically, HL-60 cells in simulated microgravity for 6, 19, 42, 47, and 49 d had 3.8-fold fewer cells that were able to participate in respiratory burst activity than cells from 1×g cultures (P=0.0011, N=5). The quantity of respiratory burst products from the cells in simulated microgravity was also significantly reduced. The fold increase over controls in mean fluorescence intensities for oxidative products from cells in microgravity was 1.1±0.1 versus 1.8±0.3 for cells at 1 ×g (P=0.013, N=4). Furthermore, the kinetic response for phorbol ester-stimulated burst activity was affected by simulated microgravity. These results demonstrate that simulated microgravity alters an innate cellular function (burst activity). If respiratory burst activity is impaired by true microgravity, then recovery from infections during spaceflight could be delayed. Finally, RWVs provide an excellent model for investigating the mechanisms associated with microgravity-induced changes in nonadherent cells.