Oxidative Metabolism Of Polymorphonuclear Leukocytes Research Articles

Differential effects of acute morphine administrations on polymorphonuclear cell metabolism in various mouse strains

This paper shows that an acute morphine treatment dose-depently alters the energetic and oxidative metabolism of polymorphonuclear leukocytes obtained from BALB/c and DBA/2 mice, while phagocytic cells from C57BL/6 were not affected. In sensitive mouse strains, i.e. BALB/c and DBA/2, morphine decreased both ATP concentration and energy charge potential. At the same time, ATP catabolic products, i.e. nucleosides (inosine+adenosine) and oxypurines (hypoxanthine+xanthine+uric acid), significantly increased, indicating an imbalance between energy production and consumption. Morphine treatment also induced malondialdehyde and Superoxide anions production in leukocyte cells from sensitive mice. The opiate antagonist naloxone blocked morphine-induced modifications by the lower morphine dose. The same parameters in cells from C57BL/6 mice were not affected. These findings confirm that: i) the phagocytic cells are an important target for the in vivo effects of morphine, and ii) the genotype-dependent variation influences the immunological responsiveness to opiates.

Crystal structure at 180°K of bis-3,5-diisopropylsalicylatobisdimethylsulfoxidozinc(II) and the inhibition of seizures and polymorphonuclear leukocyte chemiluminescence

Dimethylsulfoxide (DMSO) formed a ternary complex when mixed with a Zn-3,5-diisopropylsalicylate complex of unknown structure. The structure of this new ternary complex was characterized in an initial effort to understand the nature of this compound. Since the original complex is known to have anticonvulsant activity, the new ternary complex was also examined for anticonvulsant activity. The original complex was examined for inhibition of the polymorphonuclear leukocyte (PMNL) respiratory burst in an effort to mechanistically account for zinc complex mediated anticonvulsant activity. Dissolving the structurally unknown complex in DMSO gave crystals of a characterizable complex with an empirical formula C 30H 46O 8S 2Zn. Crystallographic data: P 1 , Z=2, a=8.063(1), b=12.452(2), c=17.951(2) Å, α=74.42(1), β=77.07(1), γ=89.50(1)°. The structure was refined to R=0.03, R w=0.04 for 3815 independent reflections with I > 2 σ( I). This complex is mononuclear, with two 3,5-diisopropylsalicylate ligands and two bonded DMSO ligands, Zn(II)(3,5-DIPS) 2(DMSO) 2. Zn(II) is coordinate covalently bonded to four O atoms in a strongly distorted tetrahedral arrangement. Each DMSO ligates via its sulfoxide O atom while each 3,5-diisopropylsalicylate ligand is monodentate. The non-ligating carbonyl O atom of each 3,5-DIPS is free except for an intramolecular hydrogen bond from the hydroxy group of the same ligand. Both 3,5-DIPS acid and Zn(II)(3,5-DIPS) 2(DMSO) 2 were examined for anticonvulsant activity in the Maximal Electroshock (MES) and Metrazol (MET) models of seizures and found to prevent both types of seizures. The Zn complex was qualitatively and quantitatively more effective than treatment with the free ligand. The influence of a Zn 3,5-DIPS complex and of the ligand 3,5-DIPS on PMNL oxidative metabolism was also studied to help understand the mechanism of anticonvulsant activity of these compounds. A dose-related and significant decrease in chemiluminescent (CL) response to opsonized Zymosan was observed, and the Zn complex was significantly more effective than the free ligand.It is concluded that mononuclear Zn complexes have anticonvulsant activity in Grand Mal and Petit Mal models of seizure possibly due to inhibition of the synthesis of superoxide or down-regulation of Nitric Oxide Synthase in activated phagocytic cells of the central nervous system.

The effect in vitro of high-density lipoprotein from healthy and infected humans on the oxidative metabolism of polymorphonuclear leukocytes.

We studied the effects in vitro of high-density lipoprotein from healthy (N-HDL) and from infected humans (AP-HDL) on the oxidative metabolism of human polymorphonuclear leukocytes (PMN). Products of the H2O2-MPO-halide system were monitored by luminol-enhanced chemiluminescence and superoxide anion formation was monitored by lucigenin-enhanced chemiluminescence during stimulation of human PMN with phorbol myristate acetate (PMA) or an opsonized stimulus (OS). The results showed that N-HDL and AP-HDL affect the oxidative metabolism of PMN in different ways. The possible role of this effect is discussed.

Oxidative Metabolism of Polymorphonuclear Leukocytes in Continuous Ambulatory Peritoneal Dialysis

To evaluate the oxidative metabolism of polymorphonuclear leukocytes (PMNL) in continuous ambulatory peritoneal dialysis (CAPD) patients compared with those of hemodialysis (HD) patients, chronic renal failure (CRF) patients not yet on dialysis, and healthy controls; and to discover factors contributing to the oxidative function in CAPD patients. Thirty-five CAPD, 22 HD, 11 CRF patients were assessed; all were free from infections at the time of examination. Thirty-one healthy volunteers served as controls. The oxidative metabolism was estimated by the production of superoxide anion, which was detected by luminol-dependent zymosan stimulated chemiluminescence (CL) with whole blood assessment. The volume of superoxide production equivalent to 1 mL of circulating blood (T-CL), that equivalent to 10(4) neutrophils (CL/N) and the velocity of superoxide production (V-CL), were measured as parameters for the oxidative function of PMNL. There were no differences in all CL parameters between CAPD and HD patients. T-CL and CL/N of dialysis patients were equal to controls but those of CRF patients were significantly decreased. V-CL of dialysis patients, as well as CRF patients, was smaller than that of controls but the difference was not significant. Among nutritional status, degree of anemia, dialytic efficacy and duration of dialysis in CAPD patients, only serum albumin concentration (Alb) correlated well to all CL parameters. Hypoalbuminemic patients (Alb < 3.6 g/dL, n = 20) had significantly decreased T-CL and CL/N compared to normoalbuminemic patients (Alb > or = 3.6 g/dL, n = 15), and decreased CL/N and V-CL compared to controls. No differences in CL parameters were observed between the patients with a history of peritonitis (n = 15) and without a history of peritonitis (n = 20). Oxidative metabolism of PMNL in CAPD patients was maintained with respect to superoxide productive volume, while the oxidative velocity was relatively impaired. Furthermore, it seems that albumin has a great influence on the oxidative metabolism of PMNL in CAPD patients.

Oxidative metabolism of neutrophilic polymorphonuclear leukocytes in experimental massive pulmonary embolism

Oxidative metabolism of neutrophilic polymorphonuclear leukocytes circulating in the blood was studied by chemiluminescent analysis and the nitroblue tetrazolium reduction test. Pronounced activation of oxidative metabolism of polymorphonuclear leukocytes was observed by the third and sixth hours of massive embolism of the pulmonary arteries. Comparison of the examined parameters of oxidative metabolism of leukocytes isolated simultaneously from venous and arterial blood indicates a delay of the most active cell fraction in pulmonary vessels during massive embolism. The transfer of functionally active leukocytes from the circulating to the marginal pool of the lungs suggests their participation in damaging the pulmonary microvascular endothelium and in increasing its permeability.

Oxidative metabolism of polymorphonuclear leukocytes (PMN) in patients with IgA nephropathy.

The production of hydrogen peroxide (H2O2) by neutrophilic polymorphonuclear leukocytes (PMN) after stimulation and the infiltration of PMN in glomeruli were determined in 20 patients with primary IgA nephropathy. The H2O2 production of PMN after the stimulation was measured with a spectrophotometer using horseradish peroxidase as substrate. The results were as follows: 1) when PMN were pretreated with cytochalasin B, H2O2 production after stimulation with heat-aggregated IgG (IgG) or serum-treated zymosan (STZ) was significantly higher in patients with IgA nephropathy than in controls, and 2) there was an increased amount of PMN localized in glomeruli in patients with IgA nephropathy using immunofluorescence of monoclonal anti-PMN antibody. It appeared that the increased renal infiltration of PMN which have a high potential for production of reactive oxygen species might induce the glomerular injuries in patients with IgA nephropathy.

Polymorphonuclear neutrophil chemiluminescence in periodontal disease

The oxidative metabolism of polymorphonuclear leukocytes (PMNs) in rapidly progressive periodontitis (RPP, n = 19), localized juvenile periodontitis (LJP, n = 10), adults periodontitis (AP, n = 10) and healthy control subjects (HS, n = 39) was compared using the luminol chemiluminescence (CL) method. Possible influences of the isolation procedure on CL were circumvented by replacing starch with Haemaccell 35 as the sedimentation agent. In all groups, CL was significantly higher with autologous serum than with normal pooled serum (NPS) and there was a significant linear relationship between the two values. Comparisons of both pooled and autologous serum between patient groups and their matched controls were not statistically significant. There was a suggestion of serum-induced defects in 2 patients and 1 control. The range of individual values within each group was very heterogeneous, probably because of the many factors that are able to influence both the production of CL and the basal levels of CL observed.

Mobiluncus species in bacterial vaginosis: aspects of pathogenesis

Mobiluncus is an anaerobic motile rod associated with bacterial vaginosis. In this work, luminol-enhanced chemiluminescence was used to study the ability of Mobiluncus spp. from the vaginas of women with bacterial vaginosis to induce, in the presence of normal adult serum, oxidative metabolism of polymorphonuclear leukocytes, which is an indirect measure of phagocytic activity. M. curtisii induced a significantly (p less than 0.05) lower response than M. mulieris, which indicates that M. curtisii escapes phagocytosis more easily. Indirect immunofluorescence assays showed IgG antibodies to M. curtisii at significantly (p less than 0.01) higher titres than to M. mulieris in women with bacterial vaginosis. The titres were higher in patients with bacterial vaginosis than in women without vaginosis and healthy men. No antibodies to Mobiluncus spp. of secretory IgA type were found in vaginal washings. These results indicate that M. curtisii is a more virulent species than M. mulieris, and agree with reports of M. curtisii found in postoperative and extragenital infections.

In vitro modulating effect of human very-low-density lipoproteins on human polymorphonuclear leukocyte oxidative metabolism and migration.

In this work, the in vitro effects of very-low-density lipoproteins (VLDL) on human polymorphonuclear leukocyte (PMN) oxidative metabolism and migration were studied. VLDL stimulated PMN superoxide generation in absence of other stimulating agents. The effect of VLDL from normotriglyceridemic subjects was more marked than with VLDL from hypertriglyceridemic subjects. VLDL reduced in a dose-dependent manner the luminol-dependent chemiluminescence of PMN stimulated by phorbol myristate acetate (PMA) and, to a lesser degree, by opsonized zymosan. This effect was observed with VLDL concentrations found in healthy and hypertriglyceridemic patients. Superoxide anion generation was also reduced by preincubation of PMN with VLDL before stimulation with PMA but not opsonized zymosan. VLDL were not cytotoxic for PMN. The above effects appear to be an intrinsic property of VLDL and might lead to reduced PMN-mediated non-specific host defences in hypertriglyceridemic subjects.

Oxidative Metabolism of Polymorphonuclear Leukocytes: Modulation by Adhesive Stimuli

Different agents such as phorbol myristate acetate (PMA), N-formyl-methionyl-leucylphenylalanine (fMet-Leu-Phe), or opsonized zymosan induced an oxidative burst in rat peritoneal polymorphonuclear leukocytes (PMNs) elicited by casein. Plastic adhesion of PMNs down-regulated superoxide (O2) release stimulated by PMA or fMet-Leu-Phe but had no effect on zymosan-induced O2 generation, indicating that the O2 forming enzyme, the NADPH oxidase, was not affected by modulation and that a common step of the transductional events induced by PMA or fMet-Leu-Phe might be involved in this regulation. We demonstrated that a differential translocation of protein kinase C (PKC) was not responsible for that modulation. PMA-induced secretion of granule content (vitamin B12-binding protein) was not susceptible to modulation, suggesting that the transductional pathways leading to O2 generation and granule secretion are partly separated. The adhesion of PMNs to different substrates (glass, plastic, albumin-, laminin-, fibronectin-, poly-lysine-, or concanavalin A-coated plastic) down-regulated to different extent superoxide release. Whether the nature of the biochemical signal induced by the diverse adhesive stimuli or a physical parameter such as binding strength was involved in this differential behavior remains to be elucidated. Since adhesiveness was dependent on the state of the cytoskeleton and O2 inducers were reported to stimulate actin polymerization, we studied the F-actin content and distribution of PMNs by using the specific fluorescent probe NBD-phallacidin and an original methodology allowing a quantitative analysis of fluorescence on both adherent and suspended cells. PMA induced a polarization of F-actin on suspended PMNs but had no effect on the intracellular distribution of F-actin in adherent PMNs. Thus, we suggest that the adhesion of PMNs induced an immobilization of F-actin, possibly correlated to the down-regulation of one of the transductional pathways involved in the NADPH oxidase activation.

Alteration of the functional effects of granulocyte-macrophage colony-stimulating factor on polymorphonuclear leukocytes by membrane-fluidizing agents

Locomotion and oxidative metabolism of polymorphonuclear leukocytes from 15 patients receiving recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined in vitro. At the end of each GM-CSF treatment course, polymorphonuclear leukocyte (PMN) chemotactic responses were suppressed and no enhancement of formyl-peptide-stimulated superoxide production was observed. The priming of PMN superoxide production normally seen after in vitro GM-CSF exposure was also blunted in these cells. By using control donor PMN, two membrane-fluidizing agents, pentoxifylline and butanol, were shown to normalize suppressed PMN chemotaxis caused by in vitro GM-CSF (1 nM) exposure. Pentoxifylline, but not butanol, also reversed the effects of in vitro GM-CSF on PMN superoxide production. When PMN obtained from six patients at the end of GM-CSF therapy were exposed to pentoxifylline in vitro, the chemotactic suppression typically observed was significantly improved. The data suggest that GM-CSF may affect PMN function via mechanisms involving membrane fluidity or cell deformability or both.

Effects of some non-steroidal anti-inflammatory drug copper complexes on polymorphonuclear leukocyte oxidative metabolism

Interaction between anti-inflammatory drugs and reactive oxygen metabolites must be considered in the course of pharmacological studies intended to develop new compounds. Effects of indomethacin, aspirin, and 3,5-diisopropylsalicylic acid (3,5-DIPS) and their copper complexes on PMNL oxidative metabolism and the evolution of an acute inflammatory reaction were studied in the rat. Experiments were performed in vitro by assessment of superoxide generation and reduction of chemiluminescence by PMNLs incubated or not (control) in medium containing various concentrations of these compounds. A dose-related decrease of these parameters was observed, however, copper complexes were found to be more effective than their parent drugs or Cu gluconate. Copper complexes were also more effective anti-inflammatory agents than their parent ligands or Cu gluconate when the volume of exudate and number of exudate PMNLs were assessed after induction of pleurisy in rats by injection of isologous serum. It is concluded that modulation of the PMNL oxidative burst by copper complexes offers an accounting for the anti-inflammatory activity of these compounds.

Inhibition of polymorphonuclear leukocyte oxidative metabolism by exogenous phospholipase C

We studied the effects of exogenous, purified phospholipase C (PLC) on neutrophil oxidative metabolism, lysosomal enzyme release and aggregation. We found that PLC inhibited O 2 − and H 2O 2 generation and oxygen consumption, but did not alter glucose oxidation via the hexose monophosphate shunt. In contrast, we found a striking stimulation of aggregation and release of the lysosomal enzymes lysozyme and β-glucuronidase. In experiments designed to further characterize the mechanism of the PLC effect on membrane activation we studied the effect of PLC on intracellular calcium concentration [Ca 2+] i and found that PLC did not interfere with the fMLP-mediated rise in [Ca 2+] i, suggesting that its inhibitory effect on the respiratory burst does not involve inhibition of early signal transduction events. In addition, we found that PLC alone results in mobilization of intracellular Ca 2+ stores, consistent with its stimulatory effect on aggregation and lysosomal enzyme release.

The effect of thermal injury on murine neutrophil oxidative metabolism.

The ability of polymorphonuclear leukocytes to kill bacteria and yeast is reflected by cellular chemiluminescence or similarly by the production of H2O2 during oxidative metabolism. With the use of flow cytometry and 2'7' dichlorodihydrofluorescein-diacetate, we determined the direct effect of thermal injury and the indirect effect of burn serum on murine polymorphonuclear leukocyte oxidative metabolism after stimulation on days 1, 5, and 10 after 25% total body surface area burn. Control or burn peritoneal leukocytes and 10% control or burn serum were incubated in vitro with 2'7' dichlorodihydrofluorescein-diacetate for 15 minutes, then stimulated with phorbol 12-myristate 13-acetate. The change in polymorphonuclear leukocyte fluorescence was calculated from fluorescence histograms before and after stimulation. The oxidative metabolism of burn polymorphonuclear leukocytes was clearly depressed on days 5 and 10 after burn injury. Control polymorphonuclear leukocytes in the presence of day 5 burn serum produced decreased levels of H2O2, returning to normal by day 10. In general, bactericidal activity is markedly depressed on days 5 and 10 after thermal injury and may be associated with increased risk of sepsis.

Oxidative metabolism of polymorphonuclear leukocytes and serum opsonic activity in chronic renal failure.

Luminol-amplified chemiluminescence was used to study the oxidative metabolism of polymorphonuclear leukocytes (PMN), in resting state and in response to opsonized zymosan, in 65 patients with different degrees of chronic renal failure (CRF) or on regular dialysis treatment (RDT). Every patient was compared on the same day with a normal subject. Furthermore, the serum opsonic activity was evaluated, cross-matching zymosan opsonized by serum from CRF-RDT patients and normals with PMN from CRF-RDT patients and normals. PMN resting chemiluminescence showed a progressive increase inversely related to the glomerular filtration rate, and it remained high in patients on RDT. Zymosan-activated chemiluminescence indicated a deficit in phagocytosis for PMN of patients with a glomerular filtration rate lower than 10 ml/min, persisting in RDT patients. The serum opsonic activity was always significantly lower in CRF and in RDT patients than in the control group; this defect was already present in patients with mild renal impairment. Our findings suggest that PMN from CRF or RDT patients have an increased reactive oxygen metabolite production in the resting state that may cause cell and tissue damage; the opsonization impairment and the decreased PMN phagocytic activity contribute to increased vulnerability to infection in these patients.

Chemiluminescence of polymorphonuclear leukocytes and whole blood during acute bacterial infection.

The purpose of this study was to follow the changes in oxidative metabolism of polymorphonuclear leukocytes (PMN) and whole blood, during and after an acute bacterial infection, in otherwise healthy individuals, with the hypothesis that the majority of the subnormal activities found at clinical investigation of PMN functions in this respect, as part of the investigation of individuals with increased susceptibility to bacterial infections, is explained by subclinical infections or consequences of recent infections. 10 patients were followed from the day of admission and up to 80 days after the acute illness. Luminol- but not lucigenin-enhanced chemiluminescence (CL) of PMN was increased during the febrile period and normalized in parallel with normalization in body temperature. Both luminol- and lucigenin-enhanced CL were enhanced in whole blood during the period of fever. Subnormal activities of luminol- or lucigenin-enhanced CL were only seen sporadically. We conclude that the oxidative metabolism of PMN, as measured by lucigenin-enhanced CL, is virtually unaffected cause of the increased luminol-enhanced CL during the acute illness is suggested to be due to the increase mobilization of myeloperoxidase.

Association between nonspecific bronchial hyperreactivity and superoxide anion production by polymorphonuclear leukocytes in chronic airflow obstruction

Inflammatory reactions are believed to be important in nonspecific bronchial hyperreactivity (BHR). To investigate the potential role for oxidant-mediated modulation of BHR, we investigated oxidative metabolism of polymorphonuclear leukocytes (PMN) from the peripheral blood in 28 nonallergic patients with chronic airflow obstruction (CAO). No difference in O2- generation was found between 14 smokers and 14 ex-smokers with CAO. A significant correlation was found between the degree of BHR and O2- generation of PMN after stimulation with 20 ng/ml phorbol myristate acetate, both in smokers (r = 0.59, p less than 0.01) and in ex-smokers (r = 0.79, p less than 0.01). The results suggest that oxygen radicals in a direct or indirect way may modulate BHR. Thus, in nonallergic patients with CAO, BHR and inflammation may be linked in a similar way as in allergic patients with asthma.

A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by Neisseria meningitidis.

Luminol-enhanced chemiluminescence (CL) was used to study the ability of various strains of Neisseria meningitidis (MC) to induce oxidative metabolism of polymorphonuclear leukocytes (PMNL); an indirect measure of phagocytic activity. To circumvent variations related to different PMNL donors, a MC serogroup X strain was used as a control for indexing the CL responses induced by other MC strains. This procedure, with pooled serum from healthy blood donors to standardize opsonising conditions, gave reproducible and comparable results, irrespective of PMNL donors. Under these conditions, there was a highly significant difference between pathogenic and non-pathogenic MC strains as regards their ability to induce CL responses (p less than 0.001). The results indicated that the differences were due partly to opsonizing antibodies, partly to other differences related to pathogenicity of tested MC strains. These differences in leukocyte/MC interaction were also confirmed by phagocytic-killing experiments. The index procedure of CL measurements may be a suitable method to study the appearance of natural immunity to MC disease, as well as the pathogenicity of particular MC strains.

Association between nonspecific bronchial hyperreactivity and superoxide anion production by polymorphonuclear leukocytes in chronic air-flow obstruction.

Inflammatory reactions are believed to be important in nonspecific bronchial hyperreactivity (BHR). To investigate the potential role for oxidant-mediated modulation of BHR, we investigated oxidative metabolism of polymorphonuclear leukocytes (PMN) from the peripheral blood in 28 nonallergic patients with chronic air-flow obstruction (CAO). No difference in O2- was found between 14 smokers and 14 ex-smokers with CAO. A significant correlation was found between the degree of BHR and O2(-)-generation of PMN after stimulation with 20 ng/ml phorbol myristate acetate, both in smokers (r = 0.59, p less than 0.01) and in ex-smokers (r = 0.79, p less than 0.01). In the light of other findings in experimental animal studies, the results suggest a direct or indirect role for O2- in the modulation of BHR. Thus, in nonallergic patients with CAO, BHR and inflammation may be linked in a similar way as in allergic patients with asthma.

Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli.

We compared the degree to which Escherichia coli phase variants which do (T1P+ E. coli) or do not (T1P- E. coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity. Unopsonized T1P+ E. coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E. coli. In the presence of serum opsonins, T1P+ E. coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E. coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min. Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity. The activity stimulated by either T1P+ E. coli or T1P-latex was susceptible to inhibition by cytochalasin B. Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN. These data indicate that T1P+ E. coli stimulate PMN oxidative metabolism more effectively than do T1P- E. coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E. coli. Furthermore, T1P-latex faithfully mimics the ability of T1P+ E. coli to stimulate PMN oxidative metabolism. Such particles may be useful in further analyses of cellular responses to T1P+ E. coli.