Reactive oxygen species activate focal adhesion kinase, paxillin and p130cas tyrosine phosphorylation in endothelial cells.

Reactive oxygen species (ROS), particularly hydroxyl radical (HO*), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 microM) and herbimycin A (0.9 microM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120-130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO*. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 microM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO*, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.

Inhibition of human neutrophil binding to hydrogen peroxide-treated endothelial cells by cAMP and hydroxyl radical scavengers.

Hydrogen peroxide (H2O2) increases adherence of human polymorphonuclear neutrophils (PMN) to cultured human umbilical vein endothelial cells (HUVEC). Catalase and HO. scavengers did not affect the increased PMN adherence to HUVEC stimulated by other compounds such as phorbol myristate acetate (PMA) and thrombin, showing that the observed effect was H2O2- and HO.-specific. This effect was inhibited by hydroxyl radicals (HO.) scavengers and not by iron-chelators that do not penetrate the cells, suggesting the involvement of intracellular HO. in the increased adherence mechanism. An increase in cAMP inhibited H2O2-induced adherence, as observed with isoproterenol, isobutylmethylxanthine, and dibutyryl-cAMP. Similarly, pentoxifylline (Ptx), an HO. scavenger that also increases cAMP, inhibited H2O2-mediated adherence but had no effect on that induced by PMA or thrombin. PKA inhibitors cancelled the Ptx-induced inhibition of H2O2-mediated adherence. However, PKA inhibitors or atrial natriuretic peptide that decreases cAMP did not increase adherence, showing that decrease in cAMP is not responsible for increased adherence. HO. scavengers did not alter the H2O2-induced reduction in cAMP levels, but did inhibit the effect of H2O2 on adherence. We conclude that HO. mediates the H2O2-induced increased in PMN adherence to HUVEC, and that the increase in cAMP that mediates PKA activation downregulates this effect.

Impairment of polymorphonuclear neutrophil function in HIV-infected patients.

Impaired polymorphonuclear neutrophil (PMN) function may contribute to the onset of certain bacterial and fungal infections and to tissue damage in human immunodeficiency virus (HIV)-infected patients. Published data on PMN function in HIV infection are controversial, possibly because most studies have involved PMNs isolated from the normal blood environment by various procedures that may modify PMN responses. We therefore used flow cytometry to study the expression of adhesion molecules at the PMN surface, actin polymerization, and the oxidative burst of whole-blood PMNs in 42 HIV-infected patients at different stages of the disease. These PMNs were activated in vivo, as shown by increased expression of the adhesion molecule CD11b/CD18, reduced L-selectin antigen expression, increased actin polymerization, and increased H2O2 production. The alterations were present in asymptomatic patients with CD4+ cell counts above 500/microliters and did not increase with progression of the disease. This PMN activation could contribute to the oxidative stress described in HIV infection. Stimulation by bacterial N-formyl peptides showed dysregulation of L-selectin shedding and decreased H2O2 production after cx vivo priming with tumor necrosis factor-alpha or interleukin-8. These latter impairments, which correlated with the decrease in CD4+ lymphocyte numbers, could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.

Effects of pentoxifylline on the adherence of polymorphonuclear neutrophils to oxidant-stimulated human endothelial cells: involvement of cyclic AMP.

Cultured human umbilical vein endothelial cells (HUVECs) treated with reactive oxygen species (ROS) show increased adherence of polymorphonuclear leukocytes (PMNs). Because pentoxifylline (PTX) is known to inhibit cell interactions, we studied PMN adherence to ROS-stimulated HUVECs pretreated with PTX. ROS were generated by the oxidation of hypoxanthine by xanthine oxidase, giving rise to superoxide anion and hydrogen peroxide. Human PMNs were then added to HUVEC monolayers. After various times, the cultures were washed and the number of adherent PMNs was estimated by measuring myeloperoxidase in the total cell homogenate. PTX inhibited adherence in a concentration-dependent manner. Moreover, the increase in intracellular cAMP content varied with the PTX concentration. Isobutylmethylxanthine (IBMX) and isoproterenol (ISO) which increase intracellular cAMP content, also inhibited the adherence of PMNs to ROS-stimulated HUVECs. We conclude that cAMP is probably involved in the intracellular regulation of ROS-mediated PMN adherence to endothelial cells.

Polymorphonuclear neutrophils from human immunodeficiency virus-infected patients show enhanced activation, diminished fMLP-induced L-selectin shedding, and an impaired oxidative burst after cytokine priming.

Impaired polymorphonuclear neutrophil (PMN) function may contribute to the onset of certain life-threatening bacterial and fungal infections in human immunodeficiency virus (HIV)-infected patients. Published data on PMN functional activity in HIV infection are controversial, possibly because most studies have involved PMNs isolated from their blood environment by means of various procedures that may differently affect surface receptor expression and thereby alter cellular responses. We therefore used flow cytometry to study the expression of adhesion molecules at the PMN surface, actin polymerization, and the oxidative burst of whole-blood polymorphonuclear neutrophils in 42 HIV-infected patients at different stages of the disease. These PMNs were activated in vivo, as demonstrated by increased expression of the adhesion molecule CD11b/CD18, reduced L-selectin antigen expression, increased actin polymerization, and increased H2O2 production. The alterations were present in asymptomatic patients with CD4+ cell counts greater than 500/microL and did not increase with the progression of the disease. Stimulation by bacterial N-formyl peptides showed dysregulation of L-selectin shedding and decreased H2O2 production after ex vivo priming with tumor necrosis factor alpha or interleukin-8 (IL-8). These latter impairments, which correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels, could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.

Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation.

We compared the effects of phorbol 12-myristate 13-acetate (PMA) and thrombin with those of nonlytic concentrations of reactive oxygen species (ROS) generated by hypoxanthine (HX)-xanthine oxidase (XO) on the adhesion properties of human umbilical cord vein endothelial cells (HUVEC) to resting polymorphonuclear neutrophils (PMN). PMN adherence to HX-XO-treated HUVEC was increased approximately twofold to 2.5-fold relative to untreated HUVEC, both immediately and after 2 hours. It was not additive to that induced by PMA or thrombin stimulation of HUVEC. ROS-induced adherence was not due to platelet-activating factor (PAF) or P-selectin expression, as it was neither antagonized by BN52021 (PAF receptor antagonist) nor inhibited by anti-P-selectin monoclonal antibody (MoAb), contrary to the increased adhesion of PMA- and thrombin-stimulated HUVEC. PMN preincubated with mannose-6-P or N-acetylneuraminic acid (sialic acid), but not mannose or galactose-6-P, showed reduced adherence to ROS-treated HUVEC, suggesting that carbohydrate molecules were expressed on the latter and served as the ligand for the PMN L-selectin. Intercellular adhesion molecule (ICAM-1), constitutively present on the surface of resting HUVEC, was involved in the PMN adherence to ROS-treated HUVEC, since this adherence was inhibited by anti-ICAM-1, anti-CD11a, anti-CD11b, and anti-CD18 MoAbs. A non-CD18, non-ICAM-1-dependent mechanism is also involved in this adherence, since effects of these MoAbs were not additive; moreover, combinations of anti-CD18 and anti-ICAM-1 MoAbs with mannose-6-P and sialic acid completely inhibited PMN adherence. The increased binding of PMN to HX-XO-exposed HUVEC observed here involved IC-AM-1, but was independent of its upregulation, and another non-ICAM-1-dependent mechanism, in which carbohydrates expressed on HUVEC recognize L-selectin on PMN.

Comparative sugar degradation by (OH). produced by the iron-driven Fenton reaction and gamma radiolysis.

We compared the attack of deoxyribose and mannitol by (OH). produced by gamma radiolysis or the iron-driven Fenton reaction. Deoxyribose (DR) oxidation by gamma radiolysis gave rise to thiobarbituric reactive substances (TBARS) with a yield of 17 mol (OH). per mole of TBARS. (OH). scavengers (benzoate, formate, and pentoxifylline) protected DR from oxidation. Mannitol was similarly oxidized by (OH). produced by gamma radiolysis, with a yield of 14 mol (OH). per mole of TBARS produced. A mixture containing both DR and mannitol gave rise to TBARS production with a yield of 9 mol (OH). per mole of TBARS superior to that of each product separately, suggesting the formation of secondary radicals responsible for the degradation of DR or mannitol. When (OH). was produced by the iron-driven Fenton reaction, DR gave rise to TBARS whereas mannitol did not. Furthermore, mannitol protected DR against degradation, apparently in the same way as desferrioxamine and diethylenetriaminepentaacetic acid, suggesting that it acts as an iron chelator. It can thus be assumed that, according to the site of (OH). production, sugar molecules are degraded as a function of their rate constant with (OH). or their iron chelating capacity.

Oxidative damage to lysozyme by the hydroxyl radical: comparative effects of scavengers.

The hydroxyl radical (OH.) is a highly-damaging reactive oxygen species, given its high reactivity and the consequent generation of secondary free radicals. This study was aimed at determining the qualitative and quantitative aspects of OH. scavenging by pentoxifylline (Ptx, a methylxanthine), uric acid and thymine on the OH.-induced alterations of a protein, lysozyme. Lysozyme was inactivated by OH. with a yield of 6.5 mol OH./mol lysozyme; moreover, SDS-PAGE showed a loss of native lysozyme (14.4 kDa), the presence of dimer and trimer aggregates and characteristic fragmentation. Tryptophan fluorescence was lost before aggregation became detectable in terms of bityrosine formation. Increasing concentrations of OH. scavengers gave increasing protection of lysozyme activity. Although all three compounds scavenge OH. with high rate constants, their effects were different: uric acid and Ptx prevented aggregation and preserved enzyme activity, whereas thymine preserved activity but did not prevent aggregation. These differences appear to be related to the formation of reducing secondary radicals, underlining the importance of this mechanism in the effects of scavengers.

Mechanism of lysozyme inactivation and degradation by iron.

The site-specific lysozyme damage by iron and by iron-catalysed oxygen radicals was investigated. A solution of purified lysozyme was inactivated by Fe(II) at pH 7.4 in phosphate buffer, as tested on cleavage of Micrococcus lysodeikticus cells; this inactivation was time- and iron concentration-dependent and was associated with a loss of tryptophan fluorescence. In addition, it was reversible at pH 4, as demonstrated by lysozyme reactivation and by the intensity of the 14.4-kD-band on SDS-PAGE. Desferal (1 mM) and Detapac (1 mM) added before iron, prevented lysozyme inactivation, while catalase (100 micrograms/ml), superoxide dismutase (100 micrograms/ml) and bovine serum albumin (100 micrograms/ml) gave about 30 to 40% protection by competing with lysozyme for iron binding. The denaturing effect of iron on lysozyme was studied in the presence of H2O2 (1 mM) and ascorbate (1 mM); under these conditions the enzyme underwent partly irreversible inactivation and degradation different to that produced by gamma radiolysis-generated .OH. Catalase almost fully protected lysozyme; in contrast, mannitol (10 mM), benzoate (10 mM), and formate (10 mM) provided no protection because of their inability to access the site at which damaging species are generated. In this system, radical species were formed in a site-specific manner, and they reacted essentially with lysozyme at the site of their formation, causing inactivation and degradation differently than the hydroxyl radical.

Effects of N-acetylcysteine on diaphragmatic function and malondialdehyde content in Escherichia coli endotoxemic rats.

We evaluated the effects of sublethal Escherichia coli endotoxemia with or without concomitant administration of N-acetylcysteine, an antioxidant agent, on diaphragmatic strength, endurance, and malondialdehyde (MDA) content in rats. One hundred ninety rats were inoculated subcutaneously on 2 successive days with 0.6 and 1.2 mg/100 g body weight of E. coli lipopolysaccharide respectively (E animals, n = 100) or saline (C group, n = 90). E and C animals were divided into two groups based on administration of endotoxin or saline alone (E group, n = 55; C group, n = 47, respectively) or endotoxin or saline plus N-acetylcysteine (1 g/kg body weight/day intraperitoneally) (E-NAC group, n = 45; C-NAC group, n = 43, respectively). Diaphragmatic strength was assessed in vivo 48 h after the first endotoxin or saline administration by measuring the transdiaphragmatic pressure (Pdl) generated during electrical stimulation of the phrenic nerves at 0.5, 10, 20, 30, 50, and 100 Hz. Endurance index was calculated as the percent ratio of Pdl generated after 30 s of phrenic stimulation at 10 Hz divided by the initial force. Diaphragmatic MDA (fluorometric technique) was measured 0, 6, 18, 30, 42, and 48 h after the first dose of endotoxin or saline. Pdl for 50 and 100 Hz was significantly reduced in Group E as compared with group C. This phenomenon was associated with a reduced endurance performance as assessed by a lower diaphragmatic endurance index in E as compared with C animals (90.9 +/- 4.2 versus 114.3 +/- 4.1 respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Gamma and pulse radiolysis study of pentoxifylline, a methylxanthine.

Pentoxifylline (Ptx) is a tri-substituted purine with anti-inflammatory properties which are thought to be due, in part, to oxygen radical scavenging. This paper reports an investigation of the reaction of Ptx with the hydroxyl radical [OH.], superoxide anion, azide radical and hydrogen peroxide generated by pulse and gamma-radiolysis, which was carried out to determine the scavenging properties of Ptx towards oxygen radicals. The superoxide anion, azide radical and hydrogen peroxide did not react with Ptx, whereas OH. reacted rapidly. In gamma-radiolysis, the action of OH. on Ptx at pH 7.4 gave rise to an end-product separated by high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry as C-8-OH-Ptx (yield 0.12 x 10(-6) mol J-1). The reaction of Ptx with OH. after pulse radiolysis at pH 7-7.4 occurred with a rate constant of (7.7 +/- 1.0) x 10(9) mol-1 s-1, forming time-dependent transient radicals. The initial spectrum (2 microseconds after the pulse) showed three maxima (310, 338 and 500 nm). A decrease in the absorbance around 500 nm and an increase around 310 nm reflected a first-order reaction, suggesting a unimolecular rearrangement. It was shown by redox titration that at least two OH-adducts were formed, one with reducing and the other with oxidizing properties. These results suggest that the reducing radical may be (C-8-OH-Ptx)(.).