The effects of arachidonic and other fatty acids on canine neutrophil metabolism

The effect of arachidonic acid on the metabolic activity and chemiluminesence of canine neutrophils was investigated to gain further insight into its role in the neutrophil metabolic burst. Arachidonic acid was found to stimulate metabolic activity and luminol-augmented chemiluminescence. The increased metabolic activity was detected by both oxygen uptake measurements and assays of hexose monophosphate shunt activity. An inhibitor of lipoxygenase and cyclooxygenase,5, 8, 11, 14-eicosatetraynoic acid prevented the hexose monophosphate shunt response to arachidonic acid. Aspirin or indomethacin, blockers of cyclooxygenase, inhibited chemiluminescence but failed to block the metabolic response to arachidonic acid. Since superoxide dismutase and 2-deoxyglucose, a blocker of glucose metabolism, inhibited the chemiluminescent response of neutrophils to arachidonic acid, it is likely that oxygen radicals produced via the hexose monophosphate shunt are required for the chemiluminescent reaction. In addition it was found that inhibition of cyclooxygenase activity blocked chemiluminescence but not the metabolic stimulation induced by sodium fluoride, suggesting that the chemiluminescence stimulated by sodium fluoride is associated with endogenous fatty acid stores. From these studies it can be concluded that arachidonic acid products of the cyclooxygenase pathway do not play a significant role in the metabolic response of neutrophils when arachidonic acid or sodium fluoride is the stimulant while the lipoxygenase pathway appears to be involved. The metabolic response is not linked to the chemical reaction that causes neutrophil, chemiluminesence, although the chemiluminescent response depends on hexose monophosphate shunt activity and presumably the oxygen radicals that ultimately result from that process.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Escherichia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occurred in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement. Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis occurring in the first 10 min. The chemiluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

Surface phagocytosis of Staphylococcus epidermidis and Escherichia coli by human neutrophils: serum requirements for opsonization and chemiluminescence

Abstract We examined the serum requirements for surface phagocytosis of Staphylococcus epidermidis and Eschericia coli and for the subsequent chemiluminescent response of human neutrophils. Substantial surface phagocytosis of S. epidermidis occured in the absence of opsonins, although the presence of 10% pooled or heat-inactivated serum significantly increased phagocytosis. There was no significant difference between these opsonins, indicating that surface phagocytosis of S. epidermidis did not require complement, Unopsonized E. coli were not as readily phagocytized as S. epidermidis (33% versus 57%). In contrast to S. epidermidis optimal phagocytosis of E. coli required complement as 10% heat inactivated donor serum (HHS) was significantly less effective as an opsonin than 10% pooled healthy donor serum (PHS). The time kinetics for phagocytosis of each organism were similar, with most of the phagocytosis iluminescent response of neutrophils produced discrepant results. Maximal chemiluminescence was observed when neutrophils were stimulated with bacteria opsonized in PHS. The response to HHS-opsonized bacteria was less, and chemiluminescence to unopsonized bacteria was only marginally higher than the control, even though there was relatively good phagocytosis. These results define the opsonic requirements for surface phagocytosis of S. epidermidis and E. coli and indicate that although complement may not be required for phagocytosis, it is necessary for generation of a maximal oxidative burst, and thus may be essential for efficient intracellular killing.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.     

Lucigenin chemiluminescence as a probe for measuring reactive oxygen species production in Escherichia coli

Addition of oxygen to whole cells of Escherichia coli suspended in the presence of the chemiluminescent probe bis-N-methylacridinium nitrate (lucigenin) resulted in a light emission increase of 200% of control. Addition of air to cells showed a chemiluminescent response far less than the response to oxygen. The redox cycling agents paraquat and menadione, which are known to increase intracellular production of O2- and H2O2, were also found to cause a measurable increase in lucigenin chemiluminescence in E. coli cells when added at concentrations of 1 and 0.1 mM, respectively. The oxygen-induced chemiluminescent response was not suppressed by extracellularly added superoxide dismutase or catalase. Further, the lucigenin-dependent chemiluminescent response of aerobically grown E. coli to oxygen was significantly greater than that of cells grown anaerobically. Heat-killed cells showed no increase in chemiluminescence on the addition of either oxygen, paraquat, or menadione. These results show that lucigenin may be used as a chemiluminescent probe to demonstrate continuous intracellular production of reactive oxygen metabolites in E. coli.     

Two distinct mechanisms for stimulation of oxygen-radical production by polymorphonuclear leucocytes.

Oxygen-radical production stimulated from rat polymorphonuclear leucocytes by either unopsonized latex particles (diameter = 1.01 microM) or chemotactic peptide (N-formyl-Met-Leu-Phe) was monitored by using luminol-dependent chemiluminescence. Azide inhibited by more than 80% the luminescence response induced by chemotactic peptide whether added before or after stimulation. However, the luminescence response to latex particles was progressively less susceptible to azide inhibition if the azide was added after the stimulus. Cytochalasin B, which was shown to abolish phagocytosis of the latex beads, also abolished the chemiluminescence response. However, the same cells showed a greatly enhanced response to chemotactic peptide. Cytochalasin B-treated cells secreted approx. 45% of total cellular myeloperoxidase in response to chemotactic peptide, but there was no detectable secretion in response to unopsonized latex particles. Microperoxidase equivalent to 20% of cellular peroxidase activity added to the cells before addition of the stimulus had no effect on the response to latex particles but increased approx. 2-fold the peak rate of chemiluminescence induced by chemotactic peptide. It was concluded that the unopsonized latex particles stimulated oxygen-radical production by the mechanism that involved endocytosis, whereas chemotactic peptide stimulated production by a mechanism that involved exocytosis of myeloperoxidase, the latter mechanism requiring an increase in intracellular free [Ca2+].     

Increased sensitivity to phagocytosis of Staphylococcus E-46 after growth in artificial media

Staphylococcus E-46, which exhibits high virulence in mice mainly due to resistance to phagocytosis, gradually lost its virulence during growth in artificial media [Heart-Infusion (HI) slants]. Staphylococcus E-46 is coagulase-negative and DNase-positive, and has a capsule-like structure, and the less-virulent derivative (LVD) strain seemed not to be changed in this respect. The bacteria which had been exposed to HI slants for more than one year (= LVD strain) became more sensitive to phagocytosis by mouse peritoneal macrophages than bacteria which were kept in a lyophilized state. The chemiluminescent response of macrophages to the LVD strain was remarkably higher than that to the original Staphylococcus E-46. When the LVD or the sera for opsonization were heated, the chemiluminescence to the LVD was as low as that to the original Staphylococcus E-46. Polyacrylamide gel electrophoresis of sonication-released membrane protein from the LVD strain showed a unique band with a molecular weight of about 40,000. The factor concerned with the virulence of Staphylococcus E-46 is discussed based on these results.     

PLATELET PHAGOCYTOSIS AND AGGREGATION

The addition of latex particles to native (no anticoagulant) or citrated human platelet-rich plasma (PRP), or to a once-washed platelet suspension causes platelet aggregation. This aggregation is associated with phagocytosis of the latex particles by the platelets and appears to be due to release of adenosine diphosphate (ADP) from the platelets. Adenosine and adenosine monophosphate, which are known to inhibit platelet aggregation induced by ADP, also block that induced by latex. These compounds do not prevent the phagocytosis of latex particles by the platelet. The addition of iodoacetate and 2,4-dinitrophenol in appropriate concentrations to the PRP, prior to the addition of the latex, blocks platelet aggregation and phagocytosis. This is also true for the chelating agent ethylenediaminetetraacetate (EDTA). Platelets left in contact with latex for a sufficient period of time show loss of their granules. Leucocytes phagocytose both latex and platelets that had themselves phagocytosed latex. It is concluded that phagocytosis of latex particles by platelets resembles that by white cells, and that in both processes metabolic changes appear to be involved.     

Chemiluminescence microscopy as a tool in biomedical research.

Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene assays, measuring enzyme activity, and the detection of ions and small molecules such as Ca2+, ATP, NO, O2- and H2O2. Along with the development of new chemiluminescent probes, significant progress has been made in techniques to measure chemiluminescence. Ultra-sensitive photometers or luminometers have become widely available and can be obtained with automatic injectors and microplate readers. In addition, imaging photon detectors have been developed that allow the imaging of chemiluminescence from gels, blots, and microplates. Imaging photon detectors have also been attached to microscopes and allow imaging of chemiluminescent probes and reporter genes in cells and tissues. Specific methods of photon collection, storage, and analysis have been developed for microscopic imaging of chemiluminescence. Two of these methods are discussed in detail. The first is a method of data storage that allows days of continuous imaging without creating oversized files. The second is a method for calibrating photon imaging microscopes using a low-light standard. Such calibration will be helpful for comparing the performance of various photon imaging systems and for comparing data obtained in different laboratories.     

A chemiluminescence assay for erythrophagocytosis

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 μl, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35–45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.     

The neutrophil response to polystyrene microspheres bearing defined surface functional groups

Luminol-induced chemiluminescence (CL) and phagocytosis by human neutrophils was studied using polystyrene microsphere latices as particulate stimuli. Chemiluminescence and phagocytosis parameters were measured for particles bearing carboxyl, hydroxyl, and amino groups, as well as for the underivatized microspheres. The kinetic curves of CL were bimodal, and curve parameters were evaluated for both the early- and late-phase responses. Significant differences were found among the particle surfaces studied. Underivatized particles elicited the greatest response, particles with the amino group stimulated PMN the least, carboxyl- and hydroxyl-group-bearing particles elicited intermediate magnitudes of response. Phagocytosis data were in good agreement with that obtained from CL measurements. These data provide further evidence in favor of the hypothesis that, in protein-free systems, hydrophobic particles are more readily phagocytosed. Additionally they demonstrate that electrostatic interactions are not a significant factor for neutrophil-particle contact.     

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila Serogroup 1

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.     

The trigger function of cAMP in regulating the chemiluminescence of macrophages exposed to ethanol

The capacity of mouse peritoneal macrophages to luminol-dependent zymosan-induced chemiluminescence was investigated. It was revealed that a short-term incubation (5-10 min) of macrophages in 0.05-0.50% ethanol resulted in a dose-dependent reduction of chemiluminescent response. Ethanol induce a sharp 2-3 fold increase in the intracellular cAMP content after a 2 minutes incubation. The temporal and concentration correlation of both the processes suggests that the inhibition of chemiluminescence may be mediated by the intracellular cAMP content.     

Biochemical research on oogenesis. Aminoacyl tRNA turns over in the 42-S particles of Xenopus laevis oocytes, but its ester bond is protected against hydrolysis

The ester bond aminoacyl tRNA is protected against hydrolysis in the 42-S particles (thesaurisomes) present in Xenopus laevis previtellogenic oocytes. Deacylation of tRNA is very slow in vitro, unless ATP is present. ATP causes a partial turnover of aminoacyl tRNA in vitro, with no detectable decrease in the overall aminoacylation level of tRNA, which remains close to 100%. tRNA in the particles turns over rapidly in vivo. Since the ester bond of aminoacyl tRNA is stabilized inside the 42-S particles, this turnover cannot be a consequence of spontaneous deacylation of tRNA, followed by reacylation by the aminoacyl-tRNA synthetases associated with the particles. We rather consider this turnover as reflecting a true metabolic activity of the particles, and a direct or indirect involvement of these particles in the oocyte's protein-synthesizing system.     

Red‐shifted emission from 1,2‐dioxetane‐based chemiluminescent reactions

Commercial chemiluminescent reagents emit across a broad portion of the electromagnetic spectrum (400–500 nm). A challenge to the use of chemiluminescence to monitor biological processes is the presence of interfering substances in the biological optical window. In the present study, longer wavelength emitting fluorophores (the organic dyes Alexa 568 and Alexa 647), and a semiconductor nanoparticle (QDOT800) were used to red‐shift the emission from commercially available 1,2‐dioxetane‐based chemiluminescent substrate reactions. By adding non‐conjugated fluorescent emitters into chemiluminescent reaction mixtures, an emission peak occurred at the predicted wavelength of the fluorescent emitter. The excitation and emission from QDOT800 was preserved in the presence of a 100 µm‐thick glass barrier separating it from the chemiluminescent reaction components. The maximum tissue phantom penetration by QDOT800 emission was 8.5 mm; in comparison, the native chemiluminescent emission at 500 nm was unable to penetrate the thinnest tissue phantom of 2.5 mm. The described method for red‐shifted emissions from chemiluminescent reactions does not require direct interaction between the chemiluminescent reaction and the fluorescent emitters. This suggests that the mechanism of chemiluminescent excitation of fluorophores and QDOT800 is not exclusive to chemiluminescence resonance energy transfer or sensitized chemiluminescence, but rather by broad energization from the native chemiluminescent emission. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic and immunological effects of endotoxin in the plaice, Pleuronectes platessa L.

In an effort to identify factors contributing to the resistance of fish to endotoxin toxicity, the metabolic effects of an intraperitoneal injection of a Boivin preparation of E. coli lipopoly-saccharide (LPS) were studied in plaice over 4 days. Significant changes were found in serum concentrations of glucose, cortisol and non-esterified fatty acids but not in total or free cholesterol. The effect of injected LPS on the phagocytic capacity of kidney neutrophils was examined because of the possible use of LPS to promote non-specific immunity. There was no enhancement of the chemiluminescent response to the phagocytosis of unopsonized bacteria by neutrophils from plaice 24 h after LPS treatment.     

Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic [Ca2+], and inhibits phagocytosis in mouse macrophages

In addition to its important role in intracellular metabolic pathways, ATP appears to function as a neurotransmitter in mammalian neurones. The extracellular effects of ATP are not restricted to neurones. We describe the effects of ATP on transmembrane fluxes of monovalent and divalent cations and on phagocytosis in the J774 mouse macrophage cell line and in mouse macrophages elicited by intraperitoneal injection of thioglycollate broth. Of all nucleotides tested, only ATP is capable of depolarizing the macrophage plasma membrane potential, promoting Na+ influx and K+ efflux, effecting an increase in intracellular free Ca2+, and inhibiting phagocytosis. Nonhydrolyzable ATP analogs had no effect on membrane permeability or phagocytosis. The effect mediated by ATP is not accompanied by an increase in membrane permeability to nucleotides, indicating that the action of ATP is restricted to the external surface of macrophages.     

Role of active forms of oxygen in inducing luminol-dependent chemiluminescence in macrophages

The luminol-dependent chemiluminescence of mouse peritoneal macrophages during phagocytosis of opsonized zymosan was studied by using specific active oxygen scavengers and metabolic inhibitors. Extracellular hydrogen peroxide and superoxide anion were shown to contribute immensely to the induction of the chemiluminescence. The role of the hydroxyl radical was rather insignificant, whereas singlet oxygen was not involved in this process. The interaction between luminol and peroxide was shown to be peroxidase-dependent. An inhibitory analysis revealed that the interaction between luminol, peroxide and superoxide anion obeyed a hybrid enzyme-free radical mechanism.     

Physicochemical and functional changes in human leukemic cell line HL-60

The recently established human promyelocytic cell line HL-60 was induced to differentiate in the present of DMSO. During this process, physiochemical, and functional changes were detected simultaneously. After exposure to DMSO for more than 1 day, the cell volume decreased and the tendency for hydrophobic interaction increased. Using a hydrophobic two-phase system in counter current distribution fashion, it was then possible to separate more mature metamyelocytes and segmented granulocytes from immature myeloblasts and promyelocytes. Increased functional maturity was reflected by increased chemiluminescence (CL) response and phagocytic activity. Using yeast particles opsonized with IgG as stimulating agent, the CL response increased already after 1 day in DMSO, in parallel with increased phagocytosis of these particles. In contrast, C3b-opsonized yeast and phorbol 12-myristate 13-acetate (PMA) did not enhance the CL response conspiquously until days 3–4. These data suggest that Fc receptor function linked to phagocytosis and the activation of oxidative metabolism develop earlier than that of C3b and PMA. The dissociation between Fc- and PMA-dependent stimulation of the oxidative metabolism may reflect different mechanisms of activation.     

Immunolocation of lipoteichoic acid on group B streptococcal surface

Abstract The effect of intraperitoneal (i.p.) infection with rat cytomegalovirus on the effector functions of peritoneal macrophages was investigated. There was an influx of polymorphonuclear leukocytes into the peritoneum on day 1 followed by an influx of macrophages on day 4. The macrophages harvested on day 4 showed enhanced levels of chemiluminescence emitted during phagocytosis of zymozan particles, and enhanced capacity to kill Staphylococcus aureus . Thereafter, the chemiluminescence level and the bactericidal capacity decreased, remaining low up to 6 months post-infection. In addition, macrophages harvested from animals on day 7 showed increased phagocytosis of sheep red blood cells.