Respiratory Burst Of Polymorphonuclear Leukocytes Research Articles

OXIDATIVE STRESS IN THE PATHOGENESIS OF PERIODONTAL DISEASE

Periodontal disease is a chronic inflammatory disease caused by gram-negative bacteria, characterized by gingival inflammation and alveolar bone resorption. In the pathogenesis of periodontal disease free radicals and oxidative stress play a significant role. Free radicals are frequently formed as metabolic by-products and their overproduction leads to cell damage and the development of oxidative stress. Antioxidants are substances that reduce the effects of free radicals and represent a specific defense that protects the organism from their harmful effects. Polymorphonuclear leukocytes (PMNL) are the main immune cells in oral tissue, protecting it from the damaging effects of bacteria. Interactions of leukocytes with bacteria initiate various defensive biochemical and physiological processes that lead to the destruction of the pathogen, but also leading to the respiratory burst in PMNL, with consequential production of free radicals and local tissues damage. Free radicals cause lipid peroxidation in a tissue, DNA and protein damage, enzyme oxidation, stimulation of pro-inflammatory cytokines. Antioxidants play an important role in the protection of oral tissues from the damaging effects of free radicals. The group of enzymatic antioxidants includes superoxide dismutase, oral peroxidase, catalase and glutathione peroxidase; while nonenzymic antioxidants include uric acid, albumin, vitamin C and glutathione. Free radicals play an important role in the pathogenesis of systemic diseases and diseases localized in the oral tissues as well. An imbalance between the production of free radicals and salivary antioxidants may trigger oxidative stress the onset of which is suggested as a basis for the development of periodontal disease.

Denitrification by cystic fibrosis pathogens – Stenotrophomonas maltophilia is dormant in sputum

ObjectiveChronic Pseudomonas aeruginosa lung infection is the most severe complication for cystic fibrosis (CF) patients. Infected endobronchial mucus of CF patients contains anaerobic zones mainly due to the respiratory burst of polymorphonuclear leukocytes. We have recently demonstrated ongoing denitrification in sputum from patients infected with P. aeruginosa. Therefore we aimed to investigate, whether the pathogenicity of several known CF pathogens is correlated to their ability to perform denitrification. MethodsWe measured denitrification with N2O microsensors in concert with anaerobic growth measurements by absorbance changes and colony counting in isolates from 32 CF patients chronically infected with the highly pathogenic bacteria P. aeruginosa, Achromobacter xylosoxidans, Burkholderia multivorans or the less pathogenic bacterium Stenotrophomonas maltophilia. Consumption of NO3− and NO2− was estimated by the Griess Assay. All isolates were assayed during 2 days of incubation in anaerobic LB broth with NO3− or NO2−. PNA FISH staining of 16S rRNA was used to estimate the amount of ribosomes per bacterial cells and thereby the in situ growth rate of S. maltophilia in sputum. ResultsSupplemental NO3− caused increased production of N2O by P. aeruginosa, A. xylosoxidans and B. multivorans and increased growth for all pathogens. Growth was, however, lowest for S. maltophilia. NO3− was metabolized by all pathogens, but only P. aeruginosa was able to remove NO2−. S. maltophilia had limited growth in sputum as seen by the weak PNA FISH staining. ConclusionsAll four pathogens were able to grow anaerobically by NO3− reduction. Denitrification as demonstrated by N2O production was, however, not found in S. maltophilia isolates. The ability to perform denitrification may contribute to the pathogenicity of the infectious isolates since complete denitrification promotes faster anaerobic growth. The inability of S. maltophilia to proliferate by denitrification and therefore grow in the anaerobic CF sputum may explain its low pathogenicity in CF patients.

The effect of platelet‐derived microparticles in stored apheresis platelet concentrates on polymorphonuclear leucocyte respiratory burst

Platelet-derived microparticles (PMPs) and other proinflammatory mediators, which are accumulated during the storage process, might induce transfusion adverse events. We hypothesized that the PMP primed polymorphonuclear neutrophil (PMN) respiratory burst after the transfusion, which could be linked to the transfusion-related acute lung injury (TRALI). The PMPs were isolated by centrifugation of the platelet-free plasma from 10 apheresis platelet concentrates (A-PLTs) at 20,000×g for 1 h. The PMPs were counted by flow cytometric analysis, followed by Western blotting, that were performed on isolated PMPs. The soluble CD40 ligand (sCD40L, sCD154) was assayed with ELISA. The priming of the formyl-Met-Leu-Phe (fMLP)-activated PMN respiratory burst was measured with the hydrogen peroxide production. The PMP counts increased by 1·7-folds after 3 days of storage. Meanwhile, sCD40L also significantly increased in PMP fraction isolated from the 3-day stored A-PLTs. Furthermore, Western blotting indicated that sCD40L was involved and concentrated in PMP. The PMPs were able to effectively prime the fMLP-activated PMN respiratory burst, which was partly inhibited by CD154 monoclonal antibody or by filtration with 0·1 μM membrane. Significant relativity was existed between the PMP counts, sCD40L and priming activity during the A-PLT storage. The platelet-derived microparticles, which carried the sCD40L, accumulated in the apheresis platelet concentrates during the 5 days of storage. They primed the fMLP-activated PMN respiration burst, which might be relative to TRALI.

P327 Accuracy of prediction rules and risk factors for mortality in patients with hospital-acquired pneumonia

Chronic Pseudomonas aeruginosa lung infection is the most severe complication for cystic fibrosis (CF) patients. Infected endobronchial mucus of CF patients contains anaerobic zones mainly due to the respiratory burst of polymorphonuclear leukocytes. We have recently demonstrated ongoing denitrification in sputum from patients infected with P. aeruginosa. Therefore we aimed to investigate, whether the pathogenicity of several known CF pathogens is correlated to their ability to perform denitrification.We measured denitrification with N2O microsensors in concert with anaerobic growth measurements by absorbance changes and colony counting in isolates from 32 CF patients chronically infected with the highly pathogenic bacteria P. aeruginosa, Achromobacter xylosoxidans, Burkholderia multivorans or the less pathogenic bacterium Stenotrophomonas maltophilia. Consumption of NO3− and NO2− was estimated by the Griess Assay. All isolates were assayed during 2 days of incubation in anaerobic LB broth with NO3− or NO2−. PNA FISH staining of 16S rRNA was used to estimate the amount of ribosomes per bacterial cells and thereby the in situ growth rate of S. maltophilia in sputum.Supplemental NO3− caused increased production of N2O by P. aeruginosa, A. xylosoxidans and B. multivorans and increased growth for all pathogens. Growth was, however, lowest for S. maltophilia. NO3− was metabolized by all pathogens, but only P. aeruginosa was able to remove NO2−. S. maltophilia had limited growth in sputum as seen by the weak PNA FISH staining.All four pathogens were able to grow anaerobically by NO3− reduction. Denitrification as demonstrated by N2O production was, however, not found in S. maltophilia isolates. The ability to perform denitrification may contribute to the pathogenicity of the infectious isolates since complete denitrification promotes faster anaerobic growth. The inability of S. maltophilia to proliferate by denitrification and therefore grow in the anaerobic CF sputum may explain its low pathogenicity in CF patients.

Seminal plasma-induced suppression of the respiratory burst of polymorphonuclear leukocytes and monocytes.

Based on recent findings indicating suppression of lymphocytic functions by seminal plasma (SP) we tested the effects of SP from men with normo- and oligozoospermia (n = 7, each) on the generation of luminol-enhanced chemiluminescence (CL) of polymorphonuclear leukocytes (PMN) and monocytes (M psi) stimulated in vitro with zymosan. We found a complete suppression of CL of PMN and M psi by undiluted SP's, 1,000-fold dilutions still induced greater than or equal to 20 percent inhibition. There was no difference between normo- and oligozoospermic men in inhibition of CL both with PMN and M psi. Protein concentrations of SP's were closely the same; all SP were free of the complement components C4 and C3c. After dialysis of SP the inhibition of PMN - and M psi - generated CL was no longer present. Our results demonstrate that SP exerts extremely potent inhibition of cells mediating nonspecific defense and/or antigen presentation. The inhibitor appears to be of low molecular weight. These findings may be important for infections acquired by the genital tract and may provide an explanation for the immunotolerance of spermatozoa in the female reproductive system.

Polymorphonuclear leucocyte respiratory burst activity correlates with serum zinc level in type 2 diabetic patients with foot ulcers

Patients with diabetes mellitus (DM) are prone to infection, in part due to phagocyte dysfunction and impaired polymorphonuclear (PMN) leucocyte superoxide generation. Another frequently mentioned factor in the pathogenesis of infection in DM patients is altered zinc status. This study aims to evaluate the association between serum zinc level and PMN respiratory burst activity in patients with type 2 DM. Thirty-nine type 2 DM patients (19 with foot ulcers) and 20 healthy controls are studied. Respiratory burst activity is evaluated at baseline and in stimulated states by a nitro blue tetrazolium (NBT) reduction test. Serum zinc level is evaluated by atomic absorption spectrophotometry. Although not statistically significant, PMNs from diabetics with foot ulcers appeared to be slightly hyperactivated at the baseline state. The NBT index was significantly lower in DM patients with foot ulcers after stimulation. Mean serum zinc level was significantly lower in diabetics with foot ulcers compared to those without foot ulcers. A significant negative correlation between serum zinc level and NBT index at baseline was seen in patients with foot ulcers, but this changed to a significant positive correlation after stimulation. These findings may be explained by PMN hyperactivity at baseline and by respiratory burst dysfunction following stimulation in diabetic patients.

Respiratory burst as a biomarker for stress responses

A module for the detection of immunotoxic events within the test system Triple-Lux to be used during spaceflights was developed. It is based on the production of reactive oxygen species within the respiratory burst during phagocytosis or after stimulation of the phagocytes with phorbol 12-myristate 13-acetate (PMA). For this purpose, luminol-dependent chemiluminescence was measured. The assays were carried out with polymorphonuclear leukocytes purified from sheep peripheral blood. The influence of hydrocortisone and Cd2+ on the respiratory burst in polymorphonuclear leukocytes was assayed. Hydrocortisone in concentrations between 10(-4) and 10(-9) mol/liter showed an immunostimulating effect after PMA treatment. An immunosuppressive effect was observed for Cd2+ in concentrations between 10(-4) and 10(-7) mol/liter. Cryoconservation, which has often been critical for primary cells, can be accomplished without any subsequent loss of function by freezing the cells in dimethyl sulfoxide-containing medium.

Synthesis and characterization of new copper– and zinc–chloroquine complexes and their activities on respiratory burst of polymorphonuclear leukocytes

The metal–chloroquine (CQ) complexes, Cu(CQ) 2Cl ( 1), Cu(CQ)(PPh 3)(NO 3) ( 2), [Cu(OAc) 2(CQ)] 2 ( 3) ZnCl 2(CQ)(H 2O) 2 ( 4), [Zn(OAc) 2(CQ)(H 2O)] 2 ( 5), were synthesized and characterized by NMR, FAB-mass, elemental analysis, and UV–Vis, EPR and IR spectroscopies. The effects of these compounds on the generation of reactive oxygen species (ROS) from human neutrophils (PMNs) were tested in the concentration range 1–100 μM and compared to that of chloroquine. The data show that the copper–chloroquine complexes 1– 3 inhibit neutrophil release of ROS in PMNs activated either by a phorbol ester or by phagocytosable particles. Both effects were dose-dependent, with an IC 50 of ∼10 μM. With the same stimulants, there was only modest inhibition of ROS generation by any of the zinc–chloroquine complexes 4– 5 at 10–100 μM. All complexes did not show significant in vitro toxicity as assayed by the trypan blue exclusion method. Our results reinforce previous observations that many metal derivatives of anti-inflammatory drugs affect neutrophil functions with higher potency than their parent ligands.

Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

Influence of PMN leukocyte-mediated pancreatic damage on the systemic immune response in severe acute pancreatitis in rats.

The outcome of severe acute pancreatitis is determined by the development of the systemic inflammatory response and subsequent multiorgan dysfunction. Using the taurocholate-induced model of acute pancreatitis in rats, we investigated the relationship between early polymorphonuclear (PMN)-mediated pancreatic tissue damage and the systemic inflammatory response. The respiratory burst of PMN leukocytes was increased in animals with acute pancreatitis and was reduced by anti-ICAM-1 antibody and oxygen radical scavenger treatment after 24 hr. In acute pancreatitis a reduced number of peripheral helper T cells was evident, most likely due to L-selectin-mediated increased lymphocyte homing. After 24 hr the CD45RC(high)/CD45RC(low) ratio of helper T cells, a critical factor in T cell-mediated disease was increased due to a reduction of regulatory CD45RC(low) cells. Only the treatment with anti-ICAM-1 mAb affected these changes, indicating that immunological changes in necrotizing pancreatitis are only in part affected by early PMN leukocyte-mediated pancreatic damage.

Strenuous, acute exercise suppresses polymorphonuclear leukocyte respiratory burst under adherence to surface-adherent platelets in men

Interaction between polymorphonuclear leukocyte (PMN) and platelets is important in the pathogenesis of thrombosis and inflammation. This study investigates how strenuous, acute exercise affects PMN oxidative burst activity under adherence to surface-adherent platelets. Thirty sedentary healthy men exercised strenuously (up to maximal oxygen consumption) on a bicycle ergometer. Before and immediately after exercise, the kinetics of oxidant production, phosphorylation of various protein kinase C (PKC) isoforms, and translocation of p47(phox) in PMNs under adherence to surface-adherent platelets were measured using fluorescence microscopy combined with computerized image analysis. Analytical results can be summarized as follows: (i) either treating the platelet with P-selectin (CD62P) and glycoprotein IIb/IIIa (CD41) antibodies or treating the PMN with beta 2-integrin (CD18) and Mac-1 (CD11b) anti-bodies and PKC zeta pseudosubstrate effectively inhibits platelet-promoted oxidant production of PMN; (ii) PMNs adhesion to surface-adherent platelets is associated with a higher amount of phospho-PKC zeta and a larger ratio of membrane to cytosolic p47(phox) than suspended PMNs; (iii) strenuous, acute exercise decreases platelet-promoted oxidant production of PMN and is accompanied by suppressed phosphorylation of PKC zeta, translocation of p47(phox), and inhibition of PKC zeta pseudosubstrate to oxidant production; (iv) no significant changes occur in PKC alpha/beta II and delta phosphorylation of adherent PMNs following this exercise. Therefore, we conclude that strenuous, acute exercise suppresses platelet-promoted oxidative burst of PMN, possibly by reducing phosphorylation of PKC zeta and translocation of the cytosolic p47(phox) to the plasma membrane, thus inhibiting the assembly and activation of NADPH oxidase in PMN.

Tetramethylpyrazine scavenges superoxide anion and decreases nitric oxide production in human polymorphonuclear leukocytes

Tetramethylpyrazine is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. By electron spin resonance spin trapping methods, effects of tetramethylpyrazine on superoxide anion and nitric oxide generated by human polymorphonuclear leukocytes were studied. During the respiratory burst of polymorphonuclear leukocytes induced by N-formylmethionyl-leucyl-phenylalanine, tetramethylpyrazine scavenges superoxide anion dose-dependently, and decreases the production of nitric oxide significantly, but shows no influence on oxygen consumption. These results suggest that the effective protection of tetramethylpyrazine against ischemic brain injury might be due to its scavenging of reactive oxygen species and regulation on nitric oxide production, and consequent prevention of peroxynitrite formation.

Activation of respiratory burst in polymorphonuclear leukocytes upon contact with stimulated T cells and inhibition by high-density lipoproteins (HDLs)

Activation of respiratory burst in polymorphonuclear leukocytes upon contact with stimulated T cells and inhibition by high-density lipoproteins (HDLs)

Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B.

Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1-3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.

Effects of 2-ethylhexanoic acid on the production of reactive oxygen species in human polymorphonuclear leukocytes in vitro

2-Ethylhexanoic acid (2-EHA), is an industrial chemical and a toxic biotransformation product of the plasticizer di(2-ethylhexyl)phthalate. Its immunological effects are unknown. 2-EHA resembles structurally C18 fatty acids, which are known activators of respiratory burst in human polymorphonuclear leukocytes (PMNL). Therefore, we exposed PMNL to 2-EHA in vitro and measured the production of reactive oxygen species (ROS) and explored the associated cellular mechanisms. 2-EHA (10–2000 μM) inhibited dose-dependently formyl-methionyl-leucyl-phenylalanine (FMLP)-induced respiratory burst in PMNL. Moreover, 2-EHA decreased oxidative burst evoked by the protein kinase C (PKC) activators, phorbol myristate acetate (PMA) and dioctanoyl-s,n-glycerol (DIC 8). 2-EHA affected neither the levels of free intracellular calcium nor inhibited PKC. The results indicate that 2-EHA inhibits activation of PMNL to produce ROS, i.e. has an immunosuppressive effect in vitro. The site of action in the PKC is after activation of this enzyme.

Determination of the respiratory burst of polymorphonuclear leukocytes by microcalorimetry

Determination of the respiratory burst of polymorphonuclear leukocytes by microcalorimetry

The roles of complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) in the regulation of the immune complex-elicited respiratory burst of polymorphonuclear leukocytes in whole blood.

The binding of immune complexes (IC) to polymorphonuclear leukocytes (PMN) and the consequent respiratory burst (RB) were investigated in whole blood cell preparations suspended in 75% human serum, using flow cytometry. Blockade of the complement receptor (CR)1 receptor sites for C3b on whole blood cells using the monoclonal antibody (mAb) 3D9 resulted in a 1.9-fold increase in the IC-elicited PMN RB after 5 min of incubation, rising to 3.1-fold after 40 min. This enhancement was not due to increased IC deposition on PMN. Blockade of CR3 abrogated the mAb 3D9-induced rise in RB activity and inhibited the IC binding to PMN in a whole blood cell preparation, with or without mAb 3D9, by approximately 40% from 15-40 min while reducing their RB over 40 min to approximately one third. Blockade of CR1 on either erythrocytes (E) or leukocytes, before mixing the populations, revealed that the potentiation of the RB by mAb 3D9 was associated with abrogation of E-CR1 function, whereas blockade of leukocyte-CR1 had a diminishing effect. Exposure to IC at high concentrations induced release of both specific and azurophilic granule contents from PMN. The latter was CR3 dependent in that blockade of the receptor inhibited the lactoferrin release by one third during 40 min of incubation. In conclusion, CR3 plays a significant role in the IC-mediated generation of an RB and release of specific granules by PMN, while CR1 on whole blood cells, primarily E CR1, restricts the IC-elicited RB in PMN. We propose that CR1 in whole blood promotes the degradation of IC-bound iC3b to C3dg, thereby rendering the IC inaccessible for binding to CR3.

Erucic Acid and Erucic Acid Anilide-induced Oxidative Burst in Human Polymorphonuclear Leukocytes

Human polymorphonuclear leukocytes (PMNL) were exposed to erucic acid or erucic acid anilide to explore their effects on the production of reactive oxygen species (ROS) and the levels of free intracellular calcium. The compounds did not change the levels of intracellular calcium, but both dose-dependently induced respiratory burst in PMNL. Maximal production of ROS by erucic acid exceeded that induced by its anilide 13-fold. A protein kinase C inhibitor, Ro 31–8220, completely inhibited erucic acid and erucic acid anilide-induced production of ROS. Neither erucic acid nor erucic, acid anilide modified FMLP-induced production of ROS. However, erucic acid (500 μM) amplified 5 nM PMA-induced ROS production 1.8-fold, but did not have this effect at a lower PMA concentration. On the contrary, erucic acid anilide inhibited PMA-induced oxidative burst, and shifted the peak ROS production induced by PMA to a later time-point. The present results show that aniline moiety modifies the effects of erucic acid on the activation of PMNL, and suggest that both erucic acid and erucic acid anilide may activate PMNL through a protein kinase C-dependent mechanism.

THE THERMAL POTENTIATION OF ACETAMINOPHEN‐INHIBITED PMN OXIDATIVE METABOLISM IN VITRO

The effect of high temperatures (39, 41, and 43 °C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0–90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39–43 °C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O−2) production induced by AM was potentiated by incubating the cells at 39 or 43 °C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.

The thermal potentiation of acetaminophen-inhibited PMN oxidative metabolism in vitro.

The effect of high temperatures (39, 41, and 43 degrees C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0-90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39-43 degrees C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O2-) production induced by AM was potentiated by incubating the cells at 39 or 43 degrees C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.