Thrombin enhances opsonized zymosan-induced chemiluminescence of neutrophils

We examined the effects of alpha-thrombin (the native enzyme) on neutrophil activation as assessed by the measurement of chemiluminescence. alpha-Thrombin in physiological concentrations (10(-9)-10(-8)M) did not induce neutrophil chemiluminescence. However, when neutrophils were coincubated with opsonized zymosan and alpha-thrombin, the chemiluminescence response to opsonized zymosan was enhanced in a concentration-dependent manner. The neutrophil chemiluminescence responses to opsonized zymosan and to opsonized zymosan plus alpha-thrombin were dependent on the generation of oxygen-derived free radicals since the chemiluminescence was inhibited by superoxide dismutase. The results indicate that thrombin per se does not induce neutrophil chemiluminescence. However, thrombin enhances the chemiluminescence response to opsonized zymosan suggesting an interaction between thrombin and complement receptors in inducing neutrophil activation. The chemiluminescence response to thrombin and opsonized zymosan is the result of oxygen-derived free radicals.     

The effect of exogenous peroxidase on the intensity of luminol-dependent macrophage chemiluminescence

The influence of exogenous horse-radish peroxidase on the capacity of mouse peritoneal macrophages to luminol-dependent chemiluminescence induced by zymosan was investigated. It was revealed that peroxidase (1-50 mg/ml) increased the chemiluminescence in a dose-dependent manner. The maximum increase of the response (4-6 times) was obtained with the enzyme concentration being 10 mg/ml. It is found that peroxidase acts as a co-oxidant of the peroxide-dependent extracellular luminol oxidation. The including of the enzyme into macrophages makes it possible to register the intracellular chemiluminescence.     

4-dimethylamino-3',4'-dimethoxychalcone downregulates iNOS expression and exerts anti-inflammatory effects

Reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. We have studied the effects of a novel superoxide scavenger, 4-dimethylamino-3', 4'-dimethoxychalcone (CH11) in macrophages and in vivo. CH11 has been shown to inhibit the chemiluminescence induced by zymosan in mouse peritoneal macrophages and the cytotoxic effects of superoxide. In the same cells, the modulation by superoxide of nitric oxide (NO) production in response to zymosan was investigated. CH11 was more effective than the membrane-permeable scavenger Tiron for inhibition of inducible nitric oxide synthase (iNOS) protein expression and nitrite production. We have shown that CH11 inhibited chemiluminescence in vivo, as well as cell migration, and eicosanoid and tumor necrosis factor-alpha (TNF-alpha) levels in the mouse air pouch injected with zymosan. This chalcone derivative also exerted anti-inflammatory effects in the carrageenan paw oedema.     

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.     

Consumption of complement and activation of human neutrophils by an artificial immune complex: polyacrylic acid-IgG-polymer

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 micrograms/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.     

Evaluation of neutrophil activity and circulating immune complexes levels in diabetic patients

We have studied chemiluminescence produced by neutrophils stimulated by opsonized zymosan in insulin dependent (IDD) and non insulin dependent (NIDD) diabetic patients. Chemiluminescence was evaluated as the integral and maximum peak, total time and time to maximum peak of the response curve to opsonized zymosan. These values were then compared with circulating immune complexes (CIC) and glucose levels. Both IDD and NIDD patients had significantly higher values of chemiluminescence and CIC than normal controls. We also observed that patients who had the highest values of CIC and chemiluminescence levels were the ones with clinical microvascular complications.     

Ethanol inhibition of the chemiluminescent response of stimulated macrophages.

The effect of ethanol on the luminol-dependent chemiluminescence and cAMP level in mice peritoneal macrophages was investigated. Ethanol was shown to inhibit the chemiluminescence of macrophages by acting both as a "trap" for active radicals and as a suppressor of the cellular functional activity. A short preincubation of macrophages with ethanol results in a dose-dependent decrease of the chemiluminescent response to the stimulatory agent (opsonized zymosan). Ethanol was also shown to induce a peakwise rise of the intracellular cAMP after a 2-min incubation. The observed effects are correlated both in time and concentration, which allows the presumption that inhibition of the functional activity of macrophages is mediated by the increase of the intracellular cAMP level.     

Ca2+ flux as an initial event in phagocytosis by rat Kupffer cells

Ca2+ uptake by monolayer cultures of rat Kupffer cells was strongly stimulated immediately after contact with phagocytosable material, e.g. zymosan particles. The intensity of the luminol-mediated chemiluminescence following zymosan addition was dependent on extracellular Ca2+; addition of the Ca2+-ionophore A 23187 did not further increase the zymosan-elicited response. The superoxide-mediated chemiluminescence was not inhibited by indomethacin but could be suppressed by compounds known to inhibit Ca2+-calmodulin-dependent reactions. Analysis of their efficiency, however, suggests that the O-2 production is mediated by Ca2+ rather than Ca2+-calmodulin and that the inhibitors exert another, e.g. membrane-directed influence.     

Beta-adrenergic receptor function and oxygen radical production in bovine pulmonary alveolar macrophages

Reactive oxygen species production by bovine pulmonary alveolar macrophages was evaluated by a chemiluminescence assay utilizing luminol and opsonized zymosan. Incubation with dobutamine (5 x 10(-8) and 5 x 10(-7) M) or isoproterenol (5 x 10(-8) and 5 x 10(-7) M) prior to zymosan challenge significantly (p less than 0.05) increased the time for chemiluminescence to begin, and significantly decreased the level of maximum chemiluminescence. The agonists' inhibitory effects on maximum chemiluminescence were significantly reduced by pre-incubation with the appropriate antagonist (atenolol at 1 x 10(-6) M for dobutamine; and propranolol at 1 x 10(-6) M for isoproterenol). Salbutamol at 1 x 10(-6) M significantly reduced the level of maximum chemiluminescence only, but did not increase the time for chemiluminescence to begin. This effect was significantly reduced by the presence of the beta 2-antagonist ICI 118,551 at 1 x 10(-6) M. The results reveal the presence of beta 1- and beta 2-adrenoceptors on bovine pulmonary alveolar macrophages, and suggest that these receptors are important in the regulation of reactive oxygen species production by these cells.     

Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 μg/ml)-pretreated PMNs than in non-pretreated ones. The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.     

Correlation between chemiluminescence response and rate of zymosan uptake by rat alveolar macrophages. Scanning electron microscope observations

The rate of particle uptake by rat alveolar macrophages (AM) exposed to zymosan (mean number of zymosan particles becoming bound to 100 cells at a fixed time interval) was determined with the aid of the scanning electron microscope (SEM). The intensity of the chemiluminescence (CL) emitted by the AM on addition of zymosan, was measured concomitantly. During the whole course of CL emission, all the particles were found to be either attached or engulfed, but not ingested by the AM. A good correlation was obtained between the time dependence of CL intensity and that of the rate of particle uptake, both reaching a peak value at about 5 min after exposure. It is therefore assumed that the CL emitted by AM exposed to zymosan reflects the attachment and engulfment stages of phagocytosis.     

Effect of vitamin E on the oxidative metabolism of macrophages

The oxidative metabolism of macrophages in vitamin E deficiency was studied on Aug-Lac strain rats. Vitamin E deficiency was shown to enhance luminol-dependent chemiluminescence of macrophages stimulated by opsonized zymosan. There was also an increase in microviscosity of macrophage membrane lipid phase, that was estimated with a fluorescent probe. The incubation of macrophages with dl-alpha-tocopherol led to the inhibition of macrophage chemiluminescence. Superoxide dismutase, glutathione peroxidase and glutathione reductase activity was not affected by vitamin E deficiency.     

Oxygen-derived free radicals producing activity and survival of activated polymorphonuclear leukocytes

Summary Activation of polymorphonuclear (PMN) leukocytes is known to generate oxygen free radicals (OFR). However the fate of activated PMN leukocytes is not known. We investigated the OFR producing (chemiluminescence) activity and the survival of the activated PMN leukocytes. The study was divided into two groups. Group I, In vivo study (n = 7): zymosan (8.4 mg/kg) was administered intravenously in the anesthetized dogs and the blood samples were collected before and after 5, 15, 30, 60 and 120 min of zymosan administration. This group represents the in vivo pre-stimulated PMN leukocytes; Group II, In vitro study (n = 7): the blood were collected from dogs and further divided into two groups. Group A (n = 7): non-stimulated, without any added zymosan and group B (n = 7): zymosan was added to stimulate PMN leukocytes. Blood samples from group A and B were also collected at various time intervals similar to in vivo studies. Oxygen free radical producing activity of PMN leukocytes was monitored by measuring luminoldependent chemiluminescence (CL). Opsonized zymosan was used to activate PMN leukocytes. The studies in which the PMN leukocytes were stimulated in in vivo, both oxygen derived free radicals and superoxide dismutase (SOD) inhibitable oxygen free radical CL decreased significantly for 60 min and tended to reach thereafter to the pre-stimulated values. The resting chemiluminescence (chemiluminescence without zymosan stimulation in the assay medium) increased significantly for 15 min reaching to pre-stimulated values at 30 min and thereafter. In in vitro studies, oxygen derived free radicals CL of pre-stimulated PMN leukocytes (Group B) was depressed for the whole duration of investigation while SOD inhibitable CL was depressed for only 60 min. There was approximately a two-fold increase in the resting CL within 5 min of PMN leukocyte activation and it remained high for the whole duration of study. The chemiluminescence of non-stimulated PMN leukocytes in vitro (group A) remained practically normal throughout the period of observation. In in vivo studies, total white blood cells (WBC) and PMN leukocyte counts decreased initially and tended to approach towards pre-stimulated values at the end of the protocol. There were no changes in these counts in in vitro studies. These results indicate that the capacity to generate OFR is decreased in the in vivo and in vitro pre-stimulated PMN leukocytes. However this activity recovers with time. This study also suggests that the activated PMN leukocytes are not destroyed.     

Use of a microtitre plate chemiluminescence reader to study surface phagocytosis by human monocytes

Human mononuclear cells were separated from freshly obtained peripheral venous blood by density centrifugation and the number of monocytes present estimated by volume spectroscopy. The mononuclear cells were then placed directly into the wells of a microtitre plate and incubated for one hour at 37°C to promote adherence of the monocytes to the plastic wells. Non-adherent cells were then removed by washing, thus avoiding the need to treat the monocytes with EDTA or other reagents during cell preparation. The time course and dynamics of the chemiluminescence response of adherent monocytes towards opsonized zymosan was similar to those seen using non-adherent cells. The ability of adherent monocyte preparations to produce chemiluminescence following incubation for varying periods with T-lymphocyte conditioned medium was investigated. The use of a microtitre plate chemiluminescence reader allows several plates to be assayed over the 24-hour period and since small numbers of cells are required, many cultures can be analysed in one experiment. This technique (Patent applied for) promises to be a powerful tool for dissecting the cellular events which occur during macrophage activation and examining the effect of various lymphokines on the ability of monocytes to produce a chemiluminescence response.     

Influence of gluten-derived fractions on chemiluminescence production by human neutrophils

The effects of gliadin and glyc-gli on leukocyte chemiluminescence response were assessed in vitro. A dose-dependent increase in chemiluminescence response of neutrophils stimulated by zymosan was observed by using gliadin at concentrations ranging between 1 and 20 μg. By increasing glyc-gli concentration, a bimodal response was observed with an enhancement up to 50 μg/ml, followed by suppressive effects, which were again dose-dependent. The possible implications of these findings in human pathology are discussed.     

Effect of rifampicin and doxycycline on the chemiluminescence of leukocytes]

The effect of rifampicin and doxycycline on spontaneous and zymosan-induced chemiluminescence of polymorphonuclear leukocytes was studied on guinea pigs. The cells were incubated in the presence of the antibiotics, washed and stimulated by zymosan. Under such conditions rifampicin in therapeutic doses of 0.1 to 10 micrograms/kg and doxycycline in a dose of 100 micrograms/kg potentiated the leukocyte chemiluminescence. Investigation of the antibiotics effect on the cells without washing failed because of the direct interference of rifampicin and doxycycline with the cell-independent stage of the chemiluminescent reaction.     

Effect of redox-modified fibrinogen on blood leukocyte functions

The influence of oxidized fibrinogen on the intensity of luminol-dependent chemilumin escence of blood leukocytes, stimulated by opsonized zymosan was studied. It was shown that the introduction of fibrinogen modified by UV-irradiation in to a suspension of cells resulted in a significant increase in the intensity of the luminol-dependent chemiluminescence of leukocytes. It was suggested that oxidized fibrinogen can influence blood leukocytes, enhancing their functional activity.