The effect of antifungal agents on the chemoluminescence of polymorphonuclear leukocytes

The intensity of the formation of active forms of oxygen by Staphylococcus-activated polymorphonuclear leukocytes (PMNL) was registered according to the level of luminol-dependent chemiluminescence. Peritoneal PMNL of rats were shown to be already in an activated state. Activation probably occurred in the process of their penetration into the abdominal cavity. For this reason, further studies of three chloral and griseofulvin derivatives with respect to their influence on the metabolic activity of phagocytes were made on PMNL of human peripheral blood. The compounds under study were found to produce a suppressive effect, which was probably linked with their influence of the enzymatic cell systems taking part on the formation of active forms of oxygen.     

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.     

A mechanism of luminol-dependent chemiluminescence of human serum in the presence of hydrogen peroxide

Comparative role of proteins of the human blood plasma in luminol-dependent induction of chemiluminescence in the presence of hydrogen peroxide was studied. It was found that the largest contribution into chemiluminescence was made by the plasma fraction with the molecular mass 250 kD. Besides the intensity of chemiluminescence proves to be proportional to hemoglobin concentration, which when added to the blood plasma entered the fractions with the molecular mass about 250 kD. It is suggested that hemoglobin producing luminescence in the blood plasma is related to haptoglobin.     

Fibrinogen association with human monocytes: evidence for constitutive expression of fibrinogen receptors and for involvement of Mac-1 (CD18, CR3) in the binding

Radiolabeled fibrinogen (Fg) specifically binds to mononuclear leukocytes (MNL) and to purified monocytes, but not to nylon-nonadherent lymphocytes. The association is rapid, Ca++-dependent and reversible. MNL containing Fg-binding monocytes had not been exposed to endotoxin (less than 4 pg/mL) during the isolation and the binding test, and Fg binding was not altered by preincubation of MNL with lipopolysaccharide. The binding of Fg was inhibited by anti-Mac-1 antibodies (OKM1). Antibodies to surface-bound Fg were able to induce luminol-dependent chemiluminescence, indicating that Fg binding sites have receptor function. Emission of a signal depended on MNL exposure to Fg, on specific, divalent antibodies, but not on the antibody Fc portion. These data show that human monocytes constitutively express specific Fg receptors and suggest that Mac-1, a member of the integrin superfamily, is involved in Fg recognition.     

The effect of low power laser radiation of blue,green, and red ranges on free radical processes in blood of rats with experimental endotoxic shock

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was induced by intraperitoneal injection of lipopolysaccharide B (25 mg/kg) (LPS). Functional activity of blood leukocytes was evaluated by the method of luminol-dependent chemiluminescence, plasma superoxide dismutase activity was determined by the nitro blue tetrazolium assay, intensity of lipid peroxidation in erythrocyte membranes was estimated by cis-parinaric acid fluorescence. It was found that the low power laser radiation significantly influenced all investigated processes, in LPS-treated and control animals. The most pronounced effects were observed in all groups of LPS-treated animals, in which the laser radiation increased all investigated parameters. At the radiation dose 0.75 J/cm² green laser was the most effective, while at the dose of 1.5 J/cm² both green and red lasers produced potent effects. Possible mechanisms of the observed phenomena are discussed.     

Defective chemiluminescence response in differentiated HL60 cells due to impaired degranulation

In the presence of dimethyl sulfoxide, the promyelocytic leukemic cell line, HL60, differentiates into apparently mature polymorphonuclear leukocytes. When correlating the superoxide production from HL60 cells with the number of phagocytozing and NBT-positive cells, no difference was observed in comparison with normal peripheral blood leukocytes. In contrast, the luminol-dependent chemiluminescence was greatly impaired in the differentiated HL60 cells. Analysis of degranulation, i.e., release of myeloperoxidase and N-acetyl-beta-glucosaminidase- and myeloperoxidase-mediated iodination by HL60 cells, suggested that the defective chemiluminescence response observed in HL60 cells may be due to impaired release of myeloperoxidase from azurophilic granulae. This may lead to impaired microbicidal activity in these cells.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

A comparative cytofluorimetric analysis of the blood leukocytes in guinea pigs exposed to the phospholipase D and antigens of the causative agent of plague]

The influence of Y. pestis phospholipase D on the physiological state of leukocytes in the blood of guinea pigs was studied in vivo by flow impulse fluorometry with the use of fluorochrome acridine orange. During the first hours of observation the intensity of leukocyte fluorescence increased due to a rise in the number of polymorphonuclear leukocytes and changes in the permeability of cell membranes. Further changes in the intensity of the fluorescence of the material under study after 24 hours of observation occurred due to the appearance of activated lymphocytes in the blood stream. The processes normalized by day 21. The reaction of blood leukocytes to phospholipase D was specific in comparison with the reaction to capsular antigen, "mouse" toxin, lipopolysaccharide and the main somatic antigen.     

Role of endogenous porphyrins in the effects of low-intensity laser radiation of the red region on free radical processes in the blood of rats under experimental endotoxic shock

The role of endogenous porphyrins in the effects of laser radiation of the red region (632.8 nm) on free radical processes in the blood of rats under endotoxic shock induced by the administration of lipopolysaccharide B (25 mg/kg) has been studied. The measurements of the functional activity of polymorphonuclear leukocytes (the method of luminol-dependent chemiluminescence), the superoxide dismutase activity of blood plasma (using nitro blue tetrazolium), and the degree of lipid oxidation of erythrocyte membranes (the method of fluorescence of cis-parinaric acid) have been carried out. It has been found that low-intensity laser radiation strongly affects all processes examined irrespective of the administration of lipopolysaccharide B. The effect of radiation was most pronounced in animals injected with the polysaccharide, the changes being dependent on the concentration of endogenous porphyrins in samples.     

Ozone-induced oxidative modification of fibrinogen: Role of the D regions

Native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing cross-linked fibrin clots under the influence of thrombin and fibrin-stabilizing factor (FXIIIa). The aim of this study was to investigate mechanisms of impairment of both the molecular structure and the spatial organization of fibrinogen under ozone-induced oxidation. FTIR analysis showed that ozone treatment of the whole fibrinogen molecule results in the growth of hydroxyl, carbonyl, and carboxyl group content. A similar analysis of fibrinogen D and E fragments isolated from the oxidized protein also revealed transformation of distinct important functional groups. In particular, a remarkable decay of N–H groups within the peptide backbone was observed along with a lowering of the content of C–H groups belonging to either the aromatic moieties or the aliphatic chain CH₂ and CH₃ units. The model experiments performed showed that the rather unexpected decay of the aliphatic CH units might be caused by the action of hydroxyl radicals, these being produced in the water solution from ozone. The observed dissimilarities in the shapes of amide I bands of the fibrinogen D and E fragments before and after ozone treatment are interpreted in terms of feasible local conformational changes affecting the secondary structure of the protein. Taken as a whole, the FTIR data suggests that the terminal D fragments of fibrinogen are markedly more susceptible to the ozone-induced oxidation than the central E fragment. The data on elastic and dynamic light scattering provide evidence that, in the presence of FXIIIa, both the unoxidized and the oxidized fibrinogen molecules bind to one another in an “end-to-end” fashion to form the flexible covalently cross-linked fibrinogen homopolymers. The γ and α polypeptide chains of the oxidized fibrinogen proved to be involved in the enzymatic cross-linking more readily than those of unaffected fibrinogen. The experimental data on fibrinogen oxidation acquired in the present study, combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some reactive oxygen species actions detrimental to the protein function.     

Role of endogenous porphyrins in the effects of low-intensity laser radiation of the red region on free radical processes in the blood of rats under experimental endotoxic shock

The role of endogenous porphyrins in the effects of laser radiation of the red region (632.8 nm) on free radical processes in the blood of rats under endotoxic shock induced by the administration of lipopolysaccharide B (25 mg/kg) has been studied. The measurements of the functional activity of polymorphonuclear leukocytes (the method of luminol-dependent chemiluminescence), the superoxide dismutase activity of blood plasma (using nitro blue tetrazolium), and the degree of lipid oxidation of erythrocyte membranes (the method of fluorescence of cis-parinaric acid) have been carried out. It has been found that low-intensity laser radiation strongly affects all processes examined irrespective of the administration of lipopolysaccha-ride B. The effect of radiation was most pronounced in animals injected with the polysaccharide, the changes being dependent on the concentration of endogenous porphyrins in samples.     

Dynamics of changes in the functional activity of blood polymorphonuclear leukocytes in reversible myocardial ischemia in dogs

Changes in peripheral blood polymorphonuclear leukocytes (PNL) functional activity in dogs during pre- and post-ischemic periods was investigated using the model of dogs venous circulation reversible disturbances in the chronic experiment and luminol-dependent chemiluminescence method. It was demonstrated that single transitory myocardial ischemia (MI) (5 min.) causes positive increase in PNLs functional activity by the 12th-14th hour of post-ischemic period. Repeated short-term MI (5 min.) was accompanied by the increase in phagocytes activity occurring 6-8 hours following the beginning of post-ischemic period, i.e. two times faster than in the case of primary ischemia. The results obtained allow the conclusion that the accumulative effect of multiple MI and the increase in PNLs functional activity as one of the reasons of cardiomyocytes injury in ischemic region cause pronounced inflammatory and necrotic myocardial changes.     

Advanced oxidation protein products (AOPP): novel uremic toxins, or components of the non-enzymatic antioxidant system of the plasma proteome?

In 1996, a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP), was detected in the plasma of chronic uremic patients. It was suggested that AOPP measure highly oxidized proteins, especially albumin. Recent data in turn appear to indicate that oxidized fibrinogen is the key molecule responsible for the AOPP reaction in the human plasma. Since fibrinogen is an acute-phase reactant, it is evident that during each episode of inflammatory response, the antioxidant capacity of the plasma is enhanced. In this context, fibrinogen can be regarded as a component of the antioxidant system of the plasma proteome. It was also demonstrated that oxidized fibrinogen is bound to apolipoprotein(a) of lipoprotein(a) via lysine binding sites. Thus, apo(a) could compete with plasminogen (and/or tissue plasminogen activator) for its binding sites of fibrin(ogen), causing inhibition of fibrinolysis, and thereby promote atherosclerosis and cardiovascular disease.     

Luminol dependent chemiluminescence and thiol group oxidation provoked by neutrophils is attributable to different oxidizing species

Luminol-dependent chemiluminescence and thiol group oxidation of glutathione and human serum albumin were measured in order to demonstrate whether the inhibition of polymorphonuclear leukocyte chemiluminescence by albumin was attributable to thiol group oxidation. We have shown that:

• 1 thiol groups on glutathione and albumin are oxidized by PMNL stimulated by soluble and phagocytic stimuli;
• 2 thiol group oxidation in albumin and glutathione did not correlate with the inhibitory effects of these substances on luminol-dependent chemiluminescence with respect to time course, magnitude, effects of known scavengers or extracellular activity. It was therefore concluded that thiol group oxidation was not the cause of albumin inhibition of luminol-dependent chemiluminescence;
• 3 a metastable oxidant was identified after PMNL activation which was capable of oxidizing thiol groups but unable to elicit chemiluminescence form luminol.

     

Upregulation of Respiratory Burst of Polymorphonuclear Leukocytes By A Bleomycin Derivative, Peplomycin

The influence of peplomycin (PLM) on the respiratory burst of peripheral blood polymorphonuclear leukocytes (PMN) was investigated. Short-term (5 min) treatment of human PMN with 0.1μg/ml to 100μg/ml of PLM increased phorbol myristate acetate (PMA)-and formyl-methionyl-leucyl-phenylalanine (FMLP)-induced luminol-dependent chemiluminescence. PMN, as well as alveolar macrophages from rabbits treated with 0.5 to 1.0 mg/kg of peplomycin per day for 5 days, generated more superoxide (O₂^-) than the cells from untreated rabbits. In both PLM-treated and untreated PMN, chemiluminescence induced by FMLP and PMA was decreased to less than 50% of the control by staurosporine, superoxide dismutase (SOD) and catalase. However, the peak intensity in PLM-untrcated PMN was decreased to about 30% of the control by genislein, while this agent induced a slight decrease in peak intensity in the PLM-treated PMN. Inositol triphosphate and diacyl glycerol levels were not clearly increased by PLM, but an increase of intracellular Ca and a shift of protein kinase C (PKC) to the membrane occurred in PMN within 1 min after PLM treatment. Western blotting revealed that the tyrosine phosphorylation of a 115 kDa protein was upregulated by 5 to 50μg/ml of PLM. While, PLM suppressed SOD activity in alveolar macrophages and PMN. These results seem to indicate that PLM increases the respiratory burst of PMN and macrophages both by way of direct PKC activation and by the upregulation of protein tyrosine phosphorylation. This increased reactive oxygen generation, together with the suppression of SOD activity seems to be tissue-impairing.     

The role of the leukocytes in the blood-vasal regulation of the blood circulation

The ability of N-formyl-methionyl-leucyl-phenylalanine-activated leukocytes to influence platelets and vessels was studied. It is shown, that of activation of leukocytes causes a vasoconstriction. The de-endothelialization of the vessels increased this effect. In addition, activated leukocytes increased the platelet aggregation. It was concluded, that activation of leukocytes can trigger thrombogenesis, angiospasm, microembolic syndrome and other disturbances of blood circulation.     

Changes in the superoxide dismutase activity during stimulation of polymorphonuclear leukocytes of peripheral blood

The functional activity of the peripheral blood polymorphonuclear leukocytes (PML) was investigated by using the method of latex-stimulated luminol-dependent chemiluminescence (CL). The CL-intensity of PML taken from patients with acute myocardial infarction (MI) was found to be 20 times higher than that of normal individuals (NI). The change in activity of endogenous antioxidative enzyme systems may account for alteration of PML CL-parameters. It was established that the initial superoxide dismutase (SOD) activity of unstimulated PML from patients with MI exceeds that of NI, and that rapid increase in intercellular SOD activity (within 30 sec.) occurs in the process of PML stimulation. It was suggested that the change of SOD activity during PML stimulation was the result in the enzyme partial proteolysis in the cells. The positive correlation between initial level of SOD activity and CL-intensity of PML was observed. The investigation of the above parameters in MI dynamic showed a gradual normalization of PML CL-response and insignificant decrease in intracellular SOD activity in case of a favourable cause of the disease. Increased SOD activity in PMLs may be one of the factors contributing to a decrease in PML functional activity in the disease dynamic.     

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.     

Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase — 4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC₅₀ = 20 μM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p < 0.01) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a “vicious circle” of neutrophil activation at the inflammatory site.     

The human promyelocytic HL60 cell line: a model of myeloid cell differentiation using dimethylsulphoxide, phorbol ester and butyrate.

The release of the reactive oxygen species that accompanies the oxidative burst was studied in HL60 cells differentiated with either dimethylsulphoxide, butyrate or phorbol myristate acetate in order to establish the extent to which differentiated cells are phenotypically similar to human neutrophils, monocytes and macrophages. When phorbol myristate acetate was used as a stimulus, the rates of superoxide production by dimethylsulphoxide and butyrate differentiated HL60 cells was not significantly different from those observed in neutrophils and monocytes isolated from normal peripheral blood. Similar results were obtained when luminol-dependent chemiluminescence was measured in the presence of horseradish peroxidase using phorbol myristate acetate as the stimulus. However, in the absence of horseradish peroxidase, the luminol-dependent chemiluminescence in the dimethylsulphoxide and butyrate-differentiated HL60 cells was significantly lower than that of the control cells isolated from human blood, reflecting the absence of myeloperoxidase in the differentiated cells. In contrast, HL60 cells differentiated by phorbol myristate acetate failed to show any increased generation of superoxide or luminol-dependent chemiluminescence upon stimulation. Impaired release of lysosomal enzymes by the chemically differentiated cells suggests impairments in the extent of differentiation resulting in cells with defective azurophilic degranulation processes. It is concluded that HL60 cells differentiated by the above agents are somewhat controversial models of promyelocyte differentiation into typical neutrophilic, monocytic and macrophage-like cells.