Extracellular Chemiluminescence Research Articles

A targetable nanogenerator of nitric oxide for light-triggered cytotoxicity.

Nanoscale-vesicles that can target pathogens are valuable for biomedical applications. In this study, a photo-responsive nanogenerator of nitric oxide (NO) comprised of a hydrophobic core of 3-trifluoromethyl-4-nitroaniline (TFNA) and a hydrophilic shell of mannosylated poly[styrene-alter-(maleic acid)] was constructed to target and kill lectin-expressing cells. The release of NO from the nanogenerator (T@P-M) was effectively induced by luminol-derived chemiluminescence (CL), leading to high-efficiency killing of Escherichia coli (E. coli) treated with T@P-M. In addition, the uptake of T@P-M by Raw 264.7 macrophages was achieved by cell surface lectin-mediated endocytosis, enabling the intracellular release of NO from the internalized T@P-M upon the induction of extracellular chemiluminescence. Because in vivo-generated CL can overcome the limited penetration of exogenous light into biological tissues, T@P-M has potential uses as a targetable photo-activatable microbicide to combat pathogens bearing lectins or residing in macrophages.

Inhibition of phospholipase A2 abrogates intracellular processing of NADPH-oxidase derived reactive oxygen species in human neutrophils

Upon activation of human neutrophils, superoxide can be produced at two cellular sites; either in the plasma membrane, giving extracellular release of oxidants, or in intracellular organelles, resulting in oxidants being retained in the cell. The involvement of phospholipase A2 (PLA2) in phorbol myristate acetate (PMA)-induced activation of the two pools of NADPH-oxidase was investigated using a variety of PLA2 inhibitors and the oxidase activity was measured by luminol/isoluminol-amplified chemiluminescence (CL). Two of the seven inhibitors were without effect, two inhibitors inhibited both intra- and extracellular ROS production equally, and three inhibitors inhibited intracellular but not extracellular CL. Using another technique to measure ROS, PHPA oxidation, we found that intracellular ROS production was unaltered with the three last inhibitors, indicating that PLA2 is not involved in the NADPH-oxidase activity per se, but in the intracellular processing of the radicals necessary for the CL reaction to take place. The PLA2 inhibitors did not abolish the activity of myeloperoxidase (MPO), an enzyme necessary for intracellular CL to occur. Instead, we suggest that these PLA2 inhibitors block heterotypic granule fusion and prohibit the colocalization of ROS and MPO needed for intracellular CL activity.

Functional changes in human peripheral neutrophils in workers with different exposure to noxious agents

Peripheral neutrophils (PMN) are a useful model to study cell activation and to evaluate the multiple stressors' interference with signalling pathways. This study aimed to evaluate the effects of different exposure to noxious agents on PMN activity by chemiluminescence assays. Two groups of office workers were recruited: peripheral whole blood cells and isolated PMNs were analyzed, performing intra- and extra-cellular chemiluminescence evaluation to study compartmental release of radical species. The exposed group shows a higher emission in resting whole blood chemiluminescence than in controls; the intracellular chemiluminescence emission in urban employees was increased, whereas the extracellular was not affected. After stimulation, the extracellular chemiluminescence was decreased in urban employees, whereas the intracellular emission did not show differences. We hypothesize that once stimulated, exposed subjects' PMNs respond to stimuli less than unexposed: PMNs could be considered indicators of chronic exposure to noxious agents.

Differentiating between intra‐ and extracellular chemiluminescence in diluted whole‐blood samples

The differentiation between extra- and intracellular production of reactive oxygen species (ROS) in whole blood was measured by luminol- and isoluminol-enhanced chemiluminescence (CL). Azide (total CL inhibition), azide + horseradish peroxidase (HRP, restoring extracellular CL), superoxide dismutase + catalase (depleting extracellular ROS) and HRP (enhancing extracellular CL) were used to modulate luminol- and isoluminol-enhanced CL (10(-6) -10(-3) m luminophores) of 125× diluted whole blood which was activated by both calcium ionophore A23187 (Ca-I) and opsonized zymosan particles (OZP) separately. Both activators stimulated intra- and extracellular production of ROS. Luminol-enhanced CL of Ca-I-activated samples detected the intracellular ROS, and with the addition of HRP detected the extracellular CL as well. CL enhanced with isoluminol in concentrations of 10(-4) m or less was mostly extracellular. There was a mixture of intra- and extracellular CL in OZP-activated samples, probably because of the ingestion of luminophore molecules. Measurement of Ca-I-activated CL enhanced with 10(-4) m luminol is recommended for the detection of intracellular ROS. The addition of HRP leads to the detection of overall ROS production while the OZP-activated system with its addition of HRP can only be used to detect overall ROS production. Ca-I-activated CL enhanced with 10(-4) m isoluminol and with addition of HRP is recommended for the detection of extracellular CL.

Interaction of Staphylococcus epidermidis from infected hip prostheses with neutrophil granulocytes.

This study focuses on the interaction of Staphylococcus epidermidis isolated from granulation tissue covering infected hip prostheses and neutrophil granulocytes. Bacterial strains isolated from normal flora were used as controls. The bacteria were well characterized with routine methods and further characterized with random amplified polymorphic DNA analyses and slime tests. Phagocytosis and chemiluminescence (CL) assays were used in the neutrophil interaction studies. The prostheses strains were ingested to a lesser extent than strains from normal flora (p < or = 0.001). There was no significant difference between the prostheses strains and the normal flora strains in terms of total CL response. However, the extracellular CL response from the neutrophils was lower in comparison with the normal flora when interacting with the prostheses strains. The results of this study support the notion that S. epidermidis strains isolated from infected hip prostheses have an enhanced capacity to resist phagocytosis and that most of these strains elicit a reduced inflammatory response, measured as the production of extracellular oxidative metabolites from the neutrophils, compared to normal flora.

Lipid peroxidation caused by oxygen radicals fromFusobacterium-stimulated neutrophils as a possible model for the emergence of periodontitis

The possible contribution of bacteria and polymorphonuclear neutrophils (PMN) to the disease process of periodontitis was evaluated. Fusobacterium nucleatum has been associated with chronic adult periodontitis. Intracellular production and extracellular release of reactive oxygen species (ROS) by PMN stimulated by fusobacteria were evaluated. To estimate the potential extracellular damage that might be caused by the ROS, the lipid peroxidation (LPO) of an exogenous phospholipid, Intralipid, was assayed. The ROS production of PMN was studied by the nitroblue tetrazolium and chemiluminescence tests. The levels of malonaldehyde (MDA) and 4-hydroxyalkenals were used to indicate LPO. Fusobacterium nucleatum strains stimulated neutrophils to produce a large amount of ROS, independently of plasma complement factors. The two strains tested induced considerable intracellular, but no extracellular chemiluminescence responses during the first hour, indicating that ROS were released into phagosomes. However an incubation period of 4 h, in the presence of the extracellular lipid resulted in a high degree of LPO, presumably caused by ROS release from the Fusobacterium-stimulated PMN. ROS production and lipid peroxidation could be counteracted by vitamin E. In periodontitis local bacteria might stimulate PMN to release ROS, which cause inflammation and destruction.

Different effects of glucose on extracellular and intracellular respiratory burst response in normal human neutrophils activated with the soluble agonist fMet-Leu-Phe.

The study evaluated the effects of glucose concentration on the extracellular and intracellular activation of the respiratory burst in fMet-Leu-Phe-stimulated normal human neutrophils. Specific extracellular respiratory burst activity was measured as superoxide dismutase (SOD)-inhibitable cytochrome c reduction. Intracellular respiratory burst activity was studied using luminol-enhanced chemiluminescence in the presence of SOD and catalase, to quench extracellular chemiluminescence activity. Myeloperoxidase (MPO) release from activated neutrophils was studied by using the guaiacol technique. The extracellular respiratory burst following activation with 1 microM fMet-Leu-Phe was significantly reduced at 15 and 25 mM D-glucose (9.5 +/- 1.0 and 8.5 +/- 0.8 nmol/10(6) cells and 10 min; P < 0.01 and P < 0.001, respectively) as compared with that at 5 mM glucose (10.3 +/- 1.0 nmol/10(6) cells and 10 min). When specifically studying the intracellular respiratory burst, no difference was found between the responses at 5, 15 or 25 mM glucose. Increasing glucose concentrations also reduced the secretion of MPO from fMet-Leu-Phe-activated neutrophils. Elevated glucose concentrations inhibit the generation of extracellularly released reactive oxygen metabolites but have no effects on the intracellular respiratory burst in fMet-Leu-Phe-stimulated normal human neutrophils.

Beta-adrenergic modulation of FMLP- and zymosan-induced intracellular and extracellular oxidant production by polymorphonuclear leukocytes.

Evaluation of catecholamine modulation of PMNL extracellular and intracellular oxidant production may reflect beneficial and harmful effects of beta-adrenergic agonists in various disease states. We investigated the kinetics and potency of adrenaline-mediated inhibition of oxidant generation in FMLP- and zymosan-stimulated PMNLs. In FMLP-stimulated cells, the short-term burst of oxidant generation was inhibited by adrenaline in a dose-dependent fashion. Intra- and extracellular chemiluminescence and extracellular superoxide anion and hydrogen peroxide generation showed similar IC50 values for adrenaline (1.3-3.0 x 10(-8) M) indicating that both extracellular and intracellular events were inhibited with the same potency. In contrast, intracellular oxidant production evoked by the phagocytosis of zymosan was only minimally affected by 3 x 10(-5) -3 x 10(-12) M adrenaline. Extracellular inhibition of oxidant production was also apparent in zymosan-stimulated cells. In conclusion, adrenaline's ability to depress extracellular generation of oxygen metabolites while retaining prolonged intracellular oxidant production for phagocytosis supports its beneficial role as selectively targeted physiological protector.

Polymorphonuclear leukocyte chemiluminescence induced by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in serum and saliva.

The ability of different strains of Actinobacillus actinomycetemcomitans (A.a.) and Haemophilus aphrophilus (H.a.) to trigger activation of an oxidative burst in human polymorphonuclear leukocytes (PMNL) was examined by measuring the luminol-amplified light emission--chemiluminescence (CL)--from these cells. Bacterial cells were incubated with PMNL from one healthy subject, in the presence of either active serum, heat-inactivated serum, saliva, or saliva and active serum. In the presence of active serum, all five H.a. strains and two out of five A.a. strains triggered a CL response. The CL induced in the presence of heat-inactivated serum was considerably less than that achieved with fresh serum. In the presence of only saliva, all strains induced considerably weaker CL responses than those induced in the presence of saliva with active serum. In the presence of serum, intracellular reactions appeared to be the main source of CL, while addition of saliva and active serum increased the extracellular CL. The results indicate that strain-dependent differences exist among A.a. strains in their ability to trigger the oxygen-dependent bactericidal mechanisms of human PNML. In contrast, the CL patterns of H.a. strains were equivalent. Various factors in the environment, such as activated complement and salivary compounds, affect the interaction of these species with neutrophils.

Protease inhibitors (gebexate mesylate and ulinastatin) stimulate intracellular chemiluminescence in human neutrophils.

The effect of protease inhibitors on the intracellular production of free radicals was investigated by measuring chemiluminescence (CL) elicited from phagocytosed luminol-bound microspheres (Lumispheres) in human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), phorbol 12-myristate 13-acetate, or diacylglycerol. Both gabexate mesylate (Foy) and ulinastatin (Miraclid), urinary trypsin inhibitor, increased intracellular CL in a dose dependent manner. Compared to control buffer without protease inhibitor, gabexate mesylate (322 micrograms/ml) caused about a 10-fold increase in intracellular CL in stimulated neutrophils, and ulinastatin (3100 U/ml) a twofold increase in neutrophils stimulated with fMLP or IL-8. When the protease inhibitors were added to the cell suspension after the phagocytosis of lumispheres, CL responses rapidly increased again to the level which was observed when both protease inhibitors and neutrophil stimulants were incubated simultaneously. In contrast, extracellular release of oxygen metabolites from stimulated neutrophils, assayed by a conventional measurement of luminol-dependent CL, was reduced by the protease inhibitors in a dose dependent fashion. When luminol-unbound microspheres were incubated with neutrophils stimulated by fMLP in luminol solution, extracellular CL was almost completely inhibited by gabexate mesylate. These results indicate that the protease inhibitors enhance the generation of intracellular CL and suppress the extracellular release of free radicals.

Luminol‐dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (greater than 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (greater than 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 mumol/l (9-fold difference), but not by calcium ionophore A23187 (15 mumol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.

The effects of N‐ethylmaleimide on extracellularly and intracellularly generated chemiluminescence in neutrophils indicate that the rate of deactivation of NADPH‐oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane

Sustained generation of reactive oxygen metabolites following respiratory burst activation in neutrophils is a result of continued replenishment of a pool of active NADPH-oxidase. The sulphydryl-modifying reagent N-ethylmaleimide (NEM) has been shown to be without effect on the turnover of activated NADPH-oxidase but to inhibit the replenishment of active oxidase molecules (Akard et al., 1988). NEM was thus used to determine the rate of deactivation of extracellularly and intracellularly generated chemiluminescence in human neutrophils. We have shown that deactivation is more rapid when activation leads to a release of oxygen metabolites (extracellular chemiluminescence) than when the metabolites are generated intracellularly. The results indicate that the rate of deactivation of NADPH-oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal membrane.

Role of myeloperoxidase in intracellular and extracellular chemiluminescence of neutrophils.

Activated polymorphonuclear leucocytes (neutrophils) can generate both intracellular and extracellular luminol dependent chemiluminescence. As luminol dependent chemiluminescence largely measures the activity of the myeloperoxidase-H2O2 system, and as the extracellular activity of this enzyme may be responsible for the tissue damage associated with inflammatory conditions such as rheumatoid arthritis, the aim of this work was to distinguish between intracellular and extracellular chemiluminescence so that the extracellular activity of this enzyme could be evaluated. Azide was used as a non-specific inhibitor of both intracellular and extracellular chemiluminescence, whereas anti-(human myeloperoxidase) IgG was used to inhibit specifically the extracellular activity of myeloperoxidase. Thus this IgG is a useful analytical tool for studying the extracellular activity of the myeloperoxidase-H2O2 system in the pathology of rheumatoid arthritis.