The Fcgamma receptor-mediated respiratory burst of rolling neutrophils to cytokine-activated, immune complex-bearing endothelial cells depends on L-selectin but not on E-selectin

Intracellular H2O2 generation, as a measure of the respiratory burst, was determined after stimulation of neutrophils by immune complex (IC)-bearing human umbilical vein endothelial cells. Under static conditions, neutrophils basically responded to the immune deposits on resting endothelial cells. The rotating shear forces of approximately 0.7 dynes/cm2, corresponding to the physiological flow in postcapillary venules, completely abolished this basal H2O2 generation. After activation of the IC-bearing endothelial layers with interleukin-1 (IL-1) or tumor necrosis factor (TNF), or both, for 4 hours, rolling adhesion of the neutrophils was induced, accompanied by considerable H2O2 production. The neutrophil respiratory burst was prominently inhibited by anti-FcgammaRIII MoAb 3G8 (72.4%), and partially by MoAb 2E1 against FcgammaRII (38.5%). Both MoAbs together inhibited the Fc-mediated H2O2 generation by 93. 4%. The respiratory burst and rolling adhesion were markedly blocked by MoAb LAM1-3 against L-selectin (91.3%), whereas the nonfunctional anti-L-selectin MoAb LAM1-14 was ineffective. F(ab)2' fragments of MoAb 7A9 against E-selectin inhibited neutrophil rolling by 98.6%, but not the respiratory burst. Moreover, rolling adhesion of neutrophils and the related oxidative burst were CD11b/CD18- independent. In summary, L-selectin has a unique auxiliary function in triggering the FcgammaR-mediated respiratory burst of rolling neutrophils to IC-bearing endothelial cells, thereby substituting CD11b/CD18 under conditions of flow.     

Fear of contagion: a response to stress?

Alterations of neutrophil functions by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. In the present study, we examined the in vitro effects of cigarette smoke on neutrophils at times and concentrations that may be encountered during smoke exposure. We measured the level of smoke exposure in the in vitro system by measuring the levels of nicotine and comparing these to levels in the oral cavity in smokers before and after smoking. We examined both the unstimulated and stimulated release of 2 oxidative burst products: superoxide (O-2) and hydrogen peroxide (H2O2). Salivary washings were collected from 7 smokers (> 1 pack/day) before smoking a cigarette. Immediately after they smoked a cigarette, a second set of washings was collected. In vitro exposure to smoke involved incubating aliquots of neutrophils in phosphate-buffered saline for 1 to 5 minutes. Nicotine and cotinine levels were quantitated using gas chromatography, with detection by electron impact mass spectrometry. Peripheral neutrophils were isolated from medically healthy non-smoking volunteers via a double-density gradient technique and incubated in vitro with whole cigarette smoke for 0 to 5 minutes. Phorbol myristate acetate (PMA; 10(-7) M) was used to stimulate half of the cell aliquots. Superoxide generation was assessed through the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. H2O2 production was assessed through the H2O2-dependent breakdown of dichlorofluorescin diacetate to its fluorophore and measured by flow cytometry. There was a marked elevation in salivary nicotine concentration from before smoking (mean: 80.8 ng) to after smoking (mean 1,685 ng/mL). In the in vitro smoke box system, there was a time-related elevation in nicotine from 1 to 5 minutes (50-->136 ng/mL). In PMA-stimulated cells exposed to smoke, there was a time-related inhibition of both superoxide and H2O2 production. However, in unstimulated cells exposed to smoke, there was a time-related increase in the release of superoxide and H2O2. A novel finding in unstimulated cells exposed to smoke was that there appeared to be 2 distinct populations of cells--one of "high" H2O2 producers and one of "low" H2O2 producers. The proportion of high H2O2 producers increased relative to smoke exposure. The relative production of H2O2 in the unstimulated high producers was comparable to PMA-stimulated cells at 5 minutes. This release of superoxide and H2O2 in unstimulated cells exposed to smoke may alter the pathogenic processes both in periodontal diseases and other systemic diseases.     

Interactions between platelets and neutrophils in essential thrombocythaemia. Effects on neutrophil chemiluminescence and superoxide anion generation

Essential thrombocythaemia (ET) is frequently associated with neutrophil and platelet dysfunction, and with increased incidence of vascular complications (thrombosis, haemorrhage). Several interactions between platelets and neutrophils have been reported, and the reciprocal actions between these cells may have an important role both in thromboregulation and in diseases such as those caused by uncontrolled neutrophil activation. In the current paper the authors studied 15 patients affected by ET and 10 normal subjects as controls. Circulating neutrophils and platelets were purified and were recombined in constant ratios (50:1, 100:1 and 200:1) and the individual platelet to neutrophil ratio. Superoxide anion (O2-) generation and luminol-enhanced chemiluminescence (CL) were studied after neutrophil stimulation with fMLP. In normal subjects both O2- generation and CL were inhibited by autologous platelets in a dose-dependent manner. In ET patients, on the contrary, platelet-dependent inhibition of O2- generation did not occur, while a dose-dependent inhibition of CL was observed. Two groups of ET patients were found: patients with neutrophil O2- generation and CL within the normal range, and patients with significantly reduced neutrophil respiratory burst. However, no differences were found between these two groups of patients in terms of platelet effects towards fMLP-stimulated neutrophils. Therefore, platelets from ET patients were not able to exert the homeostatic control towards neutrophil O2- generation shown by platelets from normal subjects, and this phenomenon may have a role in the clinical setting. In fact, O2- has been shown to be a very strong direct platelet activator, is able to inactivate nitric oxide (which is a powerful inhibitor of platelet aggregation and adhesion to endothelium), and is directly involved in neutrophil-mediated tissue damage.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H2O2) and leukotriene B4 (LTB4) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated. 2. At a concentration of 100 microM, NAC significantly (P < 0.01) suppressed the accumulation of H2O2 in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml[-1])- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 microM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 microM and above, NAC augmented significantly the level of LTB4 in the supernatants of OZ- and fMLP-stimulated neutrophils (P < 0.01 and P < 0.05, respectively) and OZ-stimulated macrophages (P < 0.05 at 10 microM, P < 0.01 at 100 microM NAC). 3. NAC (100 microM) caused a significant (P < 0.01) reduction in the quantity of measurable H2O2 when incubated with exogenous H2O2 concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB4 when incubated with exogenous LTB4. 4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H2O2 had no significant effect on LTB4 production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H2O2 to H2O and O2, caused a pronounced, statistically significant (P < 0.01) increase in the levels of LTB4 measured in the supernatants of OZ- and fMLP-stimulated neutrophils. 5. H2O2 (12.5 microM and 25 microM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB4 (P = 0.051 and P < 0.05 at 12.5 microM and 25 microM, respectively) that was inhibited by NAC (100 microM) but not by catalase (400 u ml[-1]). 6. In conclusion, the anti-oxidant drug, NAC, increases LTB4 production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H2O2. LTB4 undergoes a H2O2-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB4, suggesting that NAC may increase LTB4 production by blocking the H2O2-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.     

Effects of hydrotherapy on pressure ulcer healing

We have earlier reported a higher Fcgamma-receptor (FcgammaR)-mediated generation of reactive oxygen species, measured as luminol-enhanced chemiluminescence (CL) from peripheral neutrophils in adult periodontitis patients. The aims of this study were to confirm our previous results and to elucidate the mechanism of this phenomenon by measuring CL in parallel with the intracellular production of hydrogen peroxide, after stimulation with opsonized bacteria. To determine whether the higher CL was associated with altered responsiveness to priming, the cells were preincubated with tumor necrosis factor alpha (TNFalpha) and lipopolysaccharide (LPS). While CL was significantly higher in subjects with periodontitis, there was no difference in hydrogen peroxide production between the patients and the controls, indicating that the hyperreactivity is related to the generation of other oxygen species than H2O2 and/or to processes in the outer cell membrane. The responsiveness to priming with LPS on CL was slightly but not significantly higher in the periodontitis group, suggesting that priming could be of value for distinguishing subjects with periodontitis. When assaying intracellular production of H2O2, TNFalpha and LPS had both a priming and an activating effect. There were no significant differences between the two groups. In conclusion, this study shows a higher FcgammaR-mediated CL of peripheral neutrophils from adult patients with periodontitis, thus confirming our earlier results. The hyperreactivity seems to be related to the outer cell membrane or to oxygen species other than H2O2.     

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils

We characterized the dose response of bovine neutrophils to platelet-activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10(-10) and 10(-9) M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (> or = 10(-7) M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10(-9) M) caused secretory granule degranulation, moderate doses (> or = 10(-7) M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10(-5) M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations > or = 10(-7) M also caused up-regulation of the adhesion molecules Mac-l and L-selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10(-6) M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10(-5) M) caused an increased rate of PMA-stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA-induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up-regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus.     

Phospholipid and cholesteryl ester transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility

1. In this study, the underlying mechanism of stimulation of respiratory burst by kazinol B, a natural isoprenylated flavan, in rat neutrophils in vitro was investigated. 2. Kazinol B concentration-dependently stimulated the superoxide anion (O2*-) generation, with a lag but transient activation profile, in neutrophils but not in a cell-free system. The maximum response (13.2+/-1.4 nmol O2*- 10 min(-1) per 10(6) cells) was observed at 10 microM kazinol B. 3. Pretreatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP) significantly enhanced the O2*- generation following the subsequent stimulation of cells with kazinol B. 4. Cells pretreated with EGTA or a protein kinase inhibitor staurosporine effectively attenuated the kazinol B-induced O2*- generation. However, a p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response. 5. Kazinol B significantly stimulated [Ca2+]i elevation in neutrophils, with a lag and slow rate of rise activation profile, and this response was attenuated by a phospholipase C (PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP2 and IP3) formation with a 1 min lag time. 6. The membrane-associated PKC-alpha and PKC-theta but not PKC-iota were increased following the stimulation of neutrophils with kazinol B. It was more rapid and sensitive in the activation of PKC-theta than PKC-alpha by kazinol B. Kazinol B partially inhibited the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to the neutrophil cytosolic PKC. 7. Neither the cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt), in the presence of ethanol, nor the protein tyrosine phosphorylation were stimulated by kazinol B. In addition, the kazinol B-induced O2*- generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein. 8. Collectively, these results indicate that the stimulation of the respiratory burst by kazinol B is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Comparison of HCV RNA levels by branched DNA probe assay and by competitive polymerase chain reaction to predict effectiveness of interferon treatment for patients with chronic hepatitis C virus

Injury to the alveolar region is a hallmark of the adult respiratory distress syndrome (ARDS) whereas injury to the epithelium of the conducting airways is a characteristic of asthma. Reactive oxygen species have been implicated as mediators of lung injury in both of these conditions. We have investigated the relationship between intracellular nonprotein thiols (NPSH), and the release of the cytosolic enzyme lactate dehydrogenase (LDH), as an index of cell injury, following treatment of the human alveolar type II-like epithelial cell line (A549 cells) or the human bronchial epithelial cell line (16HBE140-) with hydrogen peroxide (H2O2). We have also assessed the protective effects of pre-incubation of both of these cells lines with H2O2 or enhancement of intracellular NPSH against H2O2-induced cell injury. Exposure of A549 and 16HBE140- cells to H2O2 (0.1 mM and 1 mM respectively for 16 h) produced the release of 40% of the total cellular LDH. H2O2 exposure produced an initial dose-dependent decrease in NPSH in A549 cells, with a subsequent increase to above control values. 16HBE140- cells also showed a dose-dependent decrease in NPSH following exposure to H2O2. Pretreatment of A549 cells with 0.1 mM H2O2 followed by subsequent exposure to H2O2 did not protect against H2O2-induced LDH release in this epithelial cell line. Pre-incubation with 2 mM N-acetylcysteine (NAC) increased NPSH but not intracellular reduced glutathione and resulted in total inhibition of H2O2-induced LDH release in both cell types. Pretreatment with reduced glutathione protected both cell types against the injurious effects of H2O2, whereas glutathione monethyl ester (GSHMEE) only partially protected A549 cells and had no effect in 16HBE140- cells. Intracellular cysteine levels were increased in both cell lines following NAC exposure but not sufficiently to account for the increase in NPSH levels. These observations raise the possibility that a critical concentration of nonprotein thiols may be necessary to protect pulmonary epithelial cells against hydrogen peroxide-induced injury.     

Influence of antimicrobial agents on bactericidal activity of bovine milk polymorphonuclear leukocytes

The influence of nine commonly used antibiotics on the respiratory burst activity of bovine milk polymorphonuclear leukocytes (PMNL) of high yielding cows was studied in vitro. Cellular oxidative activity was quantitated after preincubation with drugs at different concentrations and assayed by a PMA (12,13-phorbol myristate acetate)-induced luminol-enhanced chemiluminescence (CL) technique. All antibiotics except sulphadiazine and enrofloxacin decreased CL at the highest concentration. Enrofloxacin significantly increased CL. Oxytetracycline inhibited CL even at low doses. The decreased CL with danofloxacin and oxytetracycline was mainly induced by their color, which caused absorption of the blue light emitted by luminol. Production of superoxide radicals measured by the cytochrome c reduction assay was lowered by danofloxacin, penicillin and chloramphenicol. The decreased CL with ceftiofur was due to inhibition of myeloperoxidase (MPO) activity and to scavenging of reactive oxygen species. Interference with the MPO-H2O2-halide system was also observed with spiramycin, erythromycin and oxytetracycline, while the latter was also observed with penicillin. The stimulatory effect of enrofloxacin might be due to an improvement of the penetration of luminol into the PMNL or to a stimulation of the production of H2O2. Potentiation of the action of PMA by changing the ratio between bound and free intracellular Ca2+ might also be involved. Our results suggest that many antibiotics may affect neutrophil function at concentrations that may be found in milk immediately after intramammary treatment or at concentrations higher than those found in milk after intramammary treatment.     

Comparison of the chemiluminescence responses of bovine neutrophils to differently opsonized zymosan particles

Stimulatory effects of 6 zymosan preparations on luminol-dependent chemiluminescence (CL) responses of isolated bovine neutrophils were compared. Unopsonized zymosan particles and zymosan particles opsonized with bovine IgG1, IgG2, fresh serum, or serum from which zymosan-specific antibodies, but not complement, had been removed (C3-serum) induced strong CL responses, with nearly equal maximal peaks in the presence of extracellular Ca2+ and Mg2+, whereas the response to fetal bovine serum-opsonized zymosan particles was markedly low. Removal of extracellular divalent cations almost completely blocked the CL reaction triggered by unopsonized, IgG1-opsonized, C3-opsonized, and fetal bovine serum-opsonized zymosan particles. By contrast, no change in the respiratory burst activity induced by serum-opsonized zymosan and only partial reduction in the response to IgG2-opsonized zymosan were seen under these conditions. Further experiments were performed with 4 zymosan preparations on neutrophils isolated from 2 calves with a genetic deficiency of CD11/CD18 membrane antigens. The unopsonized zymosan-induced CL reaction was absent in these cells. A reduced, but clear, response was observed with C3-opsonized zymosan. Unexpectedly, in the absence of extracellular Ca2+ and Mg2+, serum-opsonized zymosan failed to generate the respiratory burst, whereas response to IgG2-opsonized zymosan was normal in the CD11/CD18-deficient neutrophils. These findings indicate that unopsonized zymosan may act in a divalent cation-dependent manner at the receptor for C3bi in bovine neutrophils, as it has been shown to do in the human system. In addition, it seems that IgG2-Fc receptors capable of signaling the respiratory burst in the absence of extracellular Ca2+ and Mg2+ exist on bovine neutrophils.     

Studies of skin-window exudate human neutrophils: complex patterns of adherence to serum-coated surfaces in dependence on FMLP doses

Human neutrophils were isolated both from peripheral blood (PB) and from aseptic inflammatory exudates obtained by the Senn's skin-window (SW) technique. The respiratory burst (O2- release) and the adherence to serum-coated wells of culture microplates was investigated using a simultaneous assay. Unstimulated PB resting neutrophils did not produce a significant amount of O2- and were incapable of adhering to serum-coated plastic surfaces, while unstimulated SW neutrophils showed augmented adhesion to serum-coated culture wells. SW neutrophils were primed to enhanced FMLP-dependent O2- release in response to n-formyl-methionyl-leucylphenylalanine (FMLP). Adhesion of SW neutrophils was significantly decreased by addition of low doses (10(-10)-10(-8) M) of FMLP (from 17.1% to 8.4%, P < 0.01, N = 12), while fully activating doses (> 5 x 10(-8) M) of FMLP induced a marked increase of the cell adhesion, more pronounced in SW (39.2%) than in PB cells (27.2%). Low (5 x 10(-9) M) and high (5 x 10(-7) M) FMLP doses induced morphological changes (polarization) and actin polymerization in the neutrophils from both sources. Biphasic dose-response curves of SW neutrophil adherence were observed using FMLP, but not using concanavalin A or phorbol myristate acetate as stimulatory agents. Therefore, the adherence of SW cells appears to be regulated in a complex fashion, nonlinearly dependent on the chemotactic peptide doses and specifically regulated according to the receptors involved.     

Involvement of cyclic AMP generation in the inhibition of respiratory burst by 2-phenyl-4-quinolone (YT-1) in rat neutrophils

The inhibitory effect of 2-phenyl-4-quinolone (YT-1) on respiratory burst in rat neutrophils was investigated, and the underlying mechanism of action was assessed. YT-1 caused a concentration-dependent inhibition of the rate of O2.- release from rat neutrophils in response to formylmethionyl-leucyl-phenylalanine (fMLP), but not to phorbol 12-myristate 13-acetate (PMA), with an IC50 value of 60.7+/-8.2 microM. A comparable effect was also demonstrated in the inhibition of O2 consumption. Unlike superoxide dismutase, YT-1 had no effect on O2.- generation in the xanthine-xanthine oxidase system and during dihydroxyfumaric acid autoxidation. The fMLP-induced inositol trisphosphate (IP3) formation was unaffected by YT-1. In addition, YT-1 did not affect the initial spike of [Ca2+]i, but it accelerated the rate of [Ca2+]i decline in cells in response to fMLP. YT-1 was found to have little effect on the activity of neutrophil cytosolic protein kinase C (PKC). YT-1 increased the cellular cyclic AMP level, while having no effect on the cyclic GMP level. In addition, YT-1 increased neutrophil cytosolic protein kinase A (PKA) activity, but had no direct effect on the enzyme activity of pure porcine heart PKA. When neutrophils were treated with (8R,9S,11S)-(-)-9-hydroxy-9-hexoxycarbonyl-8-methyl-2,3,9,10-tetra hydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinde n-1-one, (KT 5720), a PKA inhibitor, the inhibition of O2.- generation by YT-1, as well as by prostaglandin E1 (PGE1) and dibutyryl cyclic AMP, was attenuated effectively. YT-1 did not activate the adenylate cyclase associated with neutrophil particulate fraction but inhibited the cytosolic phosphodiesterase (PDE) activity in a concentration-dependent manner. Neutrophils treated with YT-1 had a more pronounced increase in cellular cyclic AMP level by PGE1. Moreover, the ability of PGE1 to inhibit the respiratory burst in neutrophils was greatly enhanced by YT-1. These results suggest that the increase in cellular cyclic AMP levels by YT-1 through the inhibition of PDE (probably PDE4 isoenzyme) activity is involved in its inhibition of fMLP-induced respiratory burst in rat neutrophils.     

Elevated levels of expired breath hydrogen peroxide in bronchiectasis

Airway inflammation is important in the development and progression of many lung diseases, including bronchiectasis. Activation of inflammatory cells such as neutrophils, eosinophils, and macrophages induces a respiratory burst resulting in the production of reactive oxygen species such as hydrogen peroxide (H2O2). We have measured exhaled H2O2 in patients with documented bronchiectasis and investigated whether the concentration of H2O2 is related to the disease severity, as defined by lung function. We also investigated whether the concentrations of expired H2O2 were different in bronchiectatic patients who received inhaled corticosteroids compared with steroid-na?ve patients. In 37 patients with bronchiectasis (mean age, 45 +/- 2.5 yr; FEV1, 59 +/- 3% pred), mean H2O2 concentration in exhaled breath condensate was significantly elevated as compared with the values in 25 age-matched (mean age, 42 +/- 2 yr) normal subjects (0.87 +/- 0.01 versus 0.26 +/- 0.04 microM, p < 0.001). There was a significant negative correlation between H2O2 and FEV1 (r = -0.76, p < 0.0001). Patients treated with inhaled corticosteroids had values of H2O2 similar to those of steroid-na?ve patients (0.8 +/- 0.1 versus 0.9 +/- 0.1, p > 0.05). We conclude that H2O2 is elevated in exhaled air condensate of patients with bronchiectasis and is correlated with disease severity. Measurement of H2O2 may be used as a simple noninvasive method to monitor airway inflammation and oxidative stress.     

Cobalt and desferrioxamine reveal crucial members of the oxygen sensing pathway in HepG2 cells

Cobalt and desferrioxamine, like hypoxia, stimulate the production of erythropoietin in HepG2 cells. It is believed that cobalt as well as desferrioxamine interact with the central iron atom of heme proteins by changing their redox state similar to hypoxia. A subsequent decrease of the intracellular H2O2 levels under hypoxia was presumed to be the key event for stimulating erythropoietin production. We therefore investigated whether cobalt and desferrioxamine control the intracellular H2O2 levels that regulate gene expression by interacting with hemeproteins. Deconvolution of light absorption spectra revealed respiratory heme proteins such as cytochrome c, b558 and cytochrome aa3, as well as cytochrome b558, which is a nonrespiratory heme protein found in HepG2 cells. Whereas respiratory heme proteins are located in mitochondria, cytochrome b558 similar to the one described for the neutrophil NADPH oxidase can be visualized in the cell membrane of HepG2 cells by immunohistochemistry. Incubation with cobalt (100 microM/24 hr) interacts predominantly with cytochrome b558 and cytochrome b558. The interaction of cobalt with the respiratory chain results in an increased oxygen consumption of HepG2 cells as revealed by PO2 microelectrode measurements. Desferrioxamine (130 microM/24 hr), however has no influence on the cytochromes. In response to an external application of NADH (1 mM), the membrane bound cytochrome b558 produces two times more O2- than to the external NADPH (1 mM) application. Neither desferrioxamine not cobalt has any influence on the NADH stimulated O2- generation. Incubation with cobalt or with desferrioxamine, however, leads to a decrease of the intracellular H2O2 level as revealed by the dihydrorhodamine 123 technique, perhaps causing the well-known enhanced erythropoietin production. The cobalt-induced H2O2 decrease seems to be caused by an increased activity of the glutathion peroxidase that is also induced under hypoxia. Desferrioxamine, however, leads to an apparent H2O2 decrease only because it seems to inhibit the iron catalyzed reaction of H2O2 with dihydrorhodamine 123, hinting at the occurrence of the Fenton reaction in HepG2 cells. Therefore, it must be determined whether or not degradation products of H2O2 by the Fenton reaction suppress erythropoietin production under normoxia.     

Effects of sulfite on the uptake and binding of benzo[a]pyrene diol epoxide in cultured murine respiratory epithelial cells

Sulfur dioxide (SO2) may act as a cocarcinogen with benzo[a]pyrene (BaP) in the respiratory tract. We have modeled this effect by examining the interactions of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with sulfite, the physiological form of SO2, in a murine respiratory epithelial cell line (C10). We exposed C10 cells to [3H]-anti-BPDE and determined the effects of 1 and 10 mM sulfite on the uptake and subcellular localization of labeled products. Autoradiographic analysis showed that sulfite doubled the nuclear localization of anti-BPDE-derived materials after a 4-hr incubation period. The net nuclear localization of anti-BPDE-derived materials was not affected by sulfite during the first 60 min, but nuclear localization continued to increase in the sulfite-containing incubations throughout the 4-hr incubation period. Little increase in nuclear localization of anti-BPDE-derived material was noted in the incubations without sulfite after 60 min. Subcellular fractionation was performed to determine the amount of label associated with cytosolic and nuclear fractions and to determine covalent binding to protein and DNA. Sulfite produced a modest increase in the amount of [3H]-anti-BPDE-derived products bound to protein; however, binding to nuclear DNA increased by more than 200% with 10 mM sulfite. Analysis of the supernatants from the cytosolic and nuclear fractions of cells exposed to anti-BPDE and sulfite demonstrated the presence of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10c-su lfonate (BPT-10-sulfonate). [3H]-BPT-10-sulfonate was unable to enter C10 cells, suggesting that it is formed intracellularly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Fasciola hepatica excretory-secretory products on the metabolic burst of sheep and human neutrophils

F. hepatica excretory-secretory (ES) products were found to inhibit superoxide output in phorbol myristate acetate (PMA)-stimulated sheep and human neutrophils as measured by spectrophotometry. Luminol-enhanced chemiluminescence by PMA-stimulated neutrophils from both species was again inhibited. However, nitric oxide output by PMA-stimulated human neutrophils was significantly increased in the presence of ES products, whilst in sheep it was inhibited. No major effects were noted using resting neutrophils. The results are discussed in relation to the evolution of parasite defence mechanisms.     

The MGDS examination: a systematic approach. 3. Part II of the examination: diagnosis, treatment planning, execution of treatment, maintenance and appraisal, writing-up log diaries

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.     

Resonant energy transfer from proteins to pyridine nucleotides in mitochondria

BACKGROUND: Anaesthetic agents inhibit certain functions of human neutrophils. The respiratory burst (RB) enzyme in the plasma membrane of neutrophils leads to the production of superoxide anion. The oxygen radicals are responsible for killing phagocytised micro-organisms. We investigated the in vitro influence of remifentanil, fentanyl, and alfentanil on the respiratory burst of human neutrophils. METHODS: For the flow-cytometric evaluation, leukocytes were obtained as supernatant following sedimentation and were incubated with the tested drugs. The concentrations in vitro were adjusted to conform to the plasma concentrations reported for anaesthesia and also to 10-fold higher concentrations. The RB was measured by intracellular oxidation of dihydrorhodamine to fluorescent rhodamine after induction of phorbol-myristate-acetate (PMA), Escherichia coli (E. coli) or priming by tumour necrosis factor alpha followed by stimulation of n-formyl-methionyl-leucyl-phenylalanine (TNF-alpha/FMLP). In order to exclude prestimulation of the neutrophil granulocytes, negative controls were carried out. Propidium iodide (PI) was added for viability discrimination immediately prior to flow cytometry measurement. RESULTS: Regardless of the triggering agents chosen (PMA, E. coli, TNF-alpha/FMLP), remifentanil, fentanyl, and alfentanil had no significant effect on the neutrophils' respiratory burst even in concentrations which were higher than those encountered during in vivo conditions. CONCLUSION: With respect to peri- and postoperative risk of infection, anaesthetics and analgetics with no inhibiting effect on neutrophil function should be used. These results show that remifentanil, fentanyl, and alfentanil do not influence the neutrophils' respiratory burst in vitro.     

Fibroblasts increase adhesion to neutrophils after stimulation with phorbol ester and cytokines

In our research there was spontaneous adhesion between resting fibroblasts and neutrophils in vitro which could be increased by stimulating either the coculture of cells or each cell type separately with various stimulants. Interferon-gamma, interleukin-1, and interleukin-6 significantly increased adhesion; however, the highest adhesive response was obtained when cocultures were treated with phorbol myristate acetate (PMA). As PMA-stimulated fibroblasts show the highest adhesion to resting neutrophils, it was suggested that adhesion was primarily due to an effect on fibroblasts. Without Mg2+ PMA did not stimulate fibroblast adhesion, whereas in the absence of Ca2+ the response was only partially reduced. Spontaneous adhesion was independent of both neutrophil integrins and fibroblast ICAM-1, whereas cytokine-stimulated adhesion was blocked by mAbs against ICAM-1; PMA-stimulated adhesion was not affected by mAbs anti-ICAM-1, but was partially inhibited by mAbs anti-beta 2 integrins. These results suggested the presence of mechanisms able to modulate the adhesive fibroblast-neutrophil interaction in inflammatory and wound healing processes.