Effect of zymosan-activated plasma on the deformability of rabbit polymorphonuclear leukocytes.

Intravascular infusion of inflammatory mediators causes a sudden neutropenia due to the sequestration of polymorphonuclear leukocytes (PMN) within the microvasculature of the lung and other organs. This sequestration could be due to a decrease in the ability of PMN to deform and pass through the narrow capillary bed. The purpose of this study was to determine if the complement fragments present in zymosan-activated plasma (ZAP) caused a rapid stiffening of PMN. The PMN deformability was determined by measuring the pressure required to pass PMN through a polycarbonate filter containing 5-micron pores at a constant flow rate as well as the extraction of PMN compared with red blood cells and 125I-labeled albumin by the filter. The role of the cytoskeleton in PMN deformation was examined in studies where F-actin formation was inhibited using cytochalasin B or microtubule assembly was inhibited using colchicine. The results showed that treatment with ZAP induced a rapid decrease in PMN deformability. Inhibiting the formation of F-actin made the unstimulated PMN more deformable and reduced the stiffening induced by ZAP. In contrast, inhibition of microtubule reassembly did not alter either normal deformability or the ZAP-induced decrease in deformability. In vivo, colchicine increased normal PMN margination but did not inhibit the rapid sequestration of PMN induced by infusion of ZAP. These studies indicate that ZAP induces a rapid decrease in PMN deformability that is mediated through the cytoskeleton. They suggest that this decrease is due to the polymerization of F-actin.     

Effect of anthrax toxin on the chemiluminescence of human leukocytes

The effect of substrate containing crude anthrax toxin on the phagocytosing leukocyte chemiluminescence has been studied. Preliminary toxin incubation with leukocytes for 60 min blocks cell chemiluminescence in the linear ratio effect concentration in the protein component of the toxin; the minimum concentration of the toxin protein component inhibiting the phagocytosing leukocyte luminescence is 3-5 micrograms per 5 x 10(5) cells. The substrate pure mixture of the oedema factor and protective antigen inhibits the chemiluminescence more intensively than crude toxin does. Inhibiting chemiluminescence activity of the anthrax toxin is directly proportional to its adenylate cyclase activity.     

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.     

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X_PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X_PGPC. Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X_PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X_PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X_PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X_PGPC ≤ 0.3. For X_PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.     

Mass spectrometry analysis of oxidized phospholipids

The evidence that oxidized phospholipids play a role in signaling, apoptotic events and in age-related diseases is responsible for the increasing interest for the study of this subject. Phospholipid changes induced by oxidative reactions yield a huge number of structurally different oxidation products which difficult their isolation and characterization. Mass spectrometry (MS), and tandem mass spectrometry (MS/MS) using the soft ionization methods (electrospray and matrix-assisted laser desorption ionization) is one of the finest approaches for the study of oxidized phospholipids. Product ions in tandem mass spectra of oxidized phospholipids, allow identifying changes in the fatty acyl chain and specific features such as presence of new functional groups in the molecule and their location along the fatty acyl chain. This review describes the work published on the use of mass spectrometry in identifying oxidized phospholipids from the different classes.     

Modification of proteins by isoketal-containing oxidized phospholipids

Oxidative stress frequently leads to altered function of membrane proteins. Isoketals are highly reactive products of the isoprostane pathway of free radical-induced lipid peroxidation that rapidly form covalent protein adducts and exhibit a remarkable proclivity to form protein cross links in vitro. Examination of isoketal adducts from an animal model of oxidative injury revealed that initial adducts were formed by isoketals esterified in phospholipids, representing a novel oxidative injury-associated modification of proteins by phospholipids. Maturation of adducts involved cleavage from phospholipids and conversion of adducts to a more stable chemical form that can be detected for extended periods. Because initial adducts were formed by phospholipid-esterified isoketals, the functional consequence of isoketal adduction was examined using a model membrane protein (a cardiac K(+) channel). These studies revealed that isoketal adduction profoundly altered protein function, inhibiting potassium current in a dose-dependent manner. These findings indicate that phospholipid-esterified isoketals rapidly adduct membrane proteins and that such modification can alter protein function, suggesting a generalized cellular mechanism for alteration of membrane function as a consequence of oxidative stress.     

Arachidonic acid-induced chemiluminescence of human polymorphonuclear leukocytes

When human polymorphonuclear leukocytes were incubated with arachidonic acid, a rapid light emission was observed which reached a maximum within 2 min. The magnitude of chemiluminescence depended on the number of polymorphonuclear leukocytes and the concentration of arachidonic acid. The light emission was inhibited by about 40% or 70% by 100 μM 3-amino-1-(m-(trifluromethyl)-phenyl)-2-pyrazoline (BW755C) or 100 μM nordihydroguaiaretic acid as lipoxygenase inhibitors. In contrast, 100 μM indomethacin, a cyclooxygenase inhibitor, had no effect. These results suggested a pivotal role of the lipoxygenase pathway rather than the cyclooxygenase pathway in the light emission.     

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

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

Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cels. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [³H]palmitic acid into this protein fraction is stimulated by insulin.     

Lipopolysaccharide-dependent enhancement of adherence-mediated chemiluminescence response of polymorphonuclear leukocytes

Adherence of resting polymorphonuclear leukocytes to nylon fibre increased the chemiluminescence response (CL) from 99400 to 910300 cpm/25000 PMNL. This effect could be amplified by lipopolysaccharide (LPS) priming of granulocytes in a dose-dependent fashion. The results of nylon fibre adherence experiments suggest an in vitro model that might approximate certain conditions of in vivo PMNL-endothelial adherence and respiratory burst activation, and these reactions of polymorphonuclear leukocytes may contribute to the pathomechanisms of the Adult Respiratory Distress Syndrome.     

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(～10 mg/dl; range 10-11 mg/dl), intermediate (～50 mg/dl; range 30-61 mg/dl), or high (>100 mg/dl; range 78-128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA(2) mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA(2)/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA(2) was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apoB and Lp-PLA(2)/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA(2) on apoB and Lp(a) (50-75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA(2)-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA(2) provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.     

The effect of carnosine and trolox on cellular chemiluminescence of leukocytes]

The effects of hydrophilic antioxidant carnosine, trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid), and superoxide dismutase on the myeloperoxidase activity of leukocytes, superoxide anion and active oxygen species generation have been studied. Physiological concentrations of carnosine have been shown to decrease the ability of human leukocytes to produce chemiluminescence as a result of myeloperoxidase activation. However, the chemiluminescence induced by the generation of the superoxide or its derivatives is unaffected by this process. Trolox does not inhibit the induction of superoxide-dependent chemiluminescence of leukocytes either.     

Antibodies against phospholipids and oxidized LDL in alcoholic patients

Antiphospholipid antibodies (APA) are a generic term describing antibodies that recognize various phospholipids. Hepatocyte damage is a cardinal event in the course of alcoholic liver injury and autoantibodies against phospholipids could play an important role in this process. APA in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression and they correlate significantly with disease severity. LDL oxidation is supposed to be one of the most important pathogenic mechanisms of atherosclerosis and antibodies against oxidized low-density lipoprotein (oxLDL) are some kind of an epiphenomenon of this process. The scope of our study was to determine some autoantibodies (IgG-oxLDL and antiphospholipid antibodies) and their possible changes in alcoholic patients. We studied IgG-oxLDL and four APA - anticardiolipin antibodies (ACA), antiphosphatidylserine antibodies (APSA) antiphosphatidylethanolamine antibodies (APE) and antiphosphatidylcholine antibodies (APCA) in 35 alcoholic patients with mildly affected liver function at the beginning of the abuse treatment. The control group consisted of 60 healthy blood donors. In the studied group, we obtained positive results concerning total ACA in 17.1 % of alcoholic patients (8.3 % in the control group), 11.4 % IgG-ACA (6.7 %), 8.6 % IgM-ACA (3.3 %), 14.3 % total APE (6.7 %), 14.3 % total APCA (8.3 %) and 20 % total APSA (8.3 % in the control group). The IgG-oxLDL (406.4+/-52.5 vs 499.9+/-52.5 mU/ml) was not affected in alcoholic patients. We conclude that the autoantibodies against oxLDL are present in sera of alcoholics and healthy blood donors. Based on our results which revealed a wide range of IgG-oxLDL titres in the healthy population, this parameter does not appear to be very promising for the evaluation of the risk of atherosclerosis. Alcoholics with only mild affection of liver functions did not exhibit a significantly higher prevalence of all studied antiphospholipid antibodies (ACA, APSA, APE, APCA) which could lead to membrane lesions in these patients.     

Determination of pyrrolized phospholipids in oxidized phospholipid vesicles and lipoproteins

The Ehrlich reaction was optimized to determine pyrrolized phospholipids produced as a consequence of oxidative stress. The procedure consisted of the treatment of the modified phospholipids with p-(dimethylamino)benzaldehyde at a controlled acidity temperature and the spectrophotometric determination of adducts produced. The extinction coefficient of Ehrlich adducts was calculated by using 1-[1-(2-hydroxyethyl)-1H-pyrrol-2-yl]propan-1-ol (compound 1) as standard and was 56,500 M(-1)cm(-1). The response was linear and reproducible within the range of 0.051-7.65 microM of compound 1. When the assay was applied to determination of pyrrole content in ethanolamine incubated in the presence of 0.25-1mM of 4,5(E)-epoxy-2(E)-heptenal, the complete conversion of the aldehyde into the pyrrole ring was observed and the results obtained were similar to those found when compound 1 was determined by gas chromatography. When phosphatidylethanolamine was incubated in the presence of 0.5-40 mM of 4,5(E)-epoxy-2(E)-heptenal, the phospholipid was pyrrolized similarly to ethanolamine, although there was not a quantitative conversion and the amount of pyrroles produced depended on the pH of the media. Pyrrolized phospholipids were also produced when phosphatidylethanolamine multilamellar vesicles where oxidized in the presence of either Fe(3+)/ascorbic acid or ABAP (2,2'-azobis(2-methylpropionamide) dihydrochloride) and when high-density lipoproteins were incubated in the presence of Cu(2+), thereby confirming that phospholipid pyrrolization is a common consequence of oxidative stress and that Ehrlich adducts may be valid for determining this pyrrolization.     

The lipoxygenase pathway and chemiluminescence in horse eosinophilic leukocytes

It was shown in several cell types that the dual lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid but not the cyclooxygenase inhibitor acetylsalicylic acid suppressed luminol-dependent chemiluminescence. Since lipoxygenase is known to generate chemiluminescence in vitro, these observations were interpreted as evidence for a direct contribution of the lipoxygenase pathway to light emission in intact cells. We have investigated a possible contribution of the lipoxygenase to the chemiluminescence of horse eosinophils by directly comparing the formation of the byproduct chemiluminescence with the formation of stable end-products of the lipoxygenase pathway, leukotrienes and HETEs. Azide as well as eicosatetraynoic acid almost completely inhibited chemiluminescence stimulated by the calcium ionophore A23187 but had less effect on the formation of leukotrienes. The tumour-promoting ester, phorbol myristate acetate, stimulated chemiluminescence in an azide- and eicosatetraynoic acid-sensitive manner and failed to evoke the production of leukotrienes. Azide, but also eicosatetraynoic acid inhibited the luminol-dependent chemiluminescence generated by isolated eosinophil peroxidase in the presence of H₂O₂. Our results argue against a direct role of the lipoxygenase pathway in the generation of light in horse eosinophilic leukocytes but do not exclude that product(s) of this pathway may be involved in stimulus-response coupling.