FreeRadicalBiology&Medicine, Vol. 13, pp. 21-30, 1992

0891-5849/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.

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Original Contribution HYPOXIA/REOXYGENATION STIMULATES ENDOTHELIUM TO PROMOTE NEUTROPHIL ADHESION OLIVIER PALLUY,* LUCETTE MORLIERE,* JEAN CHRISTOPHE GRIS, t CLAUDE BONNE,* and GUY MODAT* *Laboratoirede PhysiologieCellulaire, Facult6 de Pharmacie, Universit6de Montpellier I, 15 Avenue Charles Flahault, 34060 Montpellier cedex, France; and tLaboratoire d'Hrmatologie et lmmunologie, Centre Hospitalier Rrgional et Universitaire, 5 Rue Hoche, 30006 N~mescedex, France (Received 22 August 1991; Revised 19 December 1991; Accepted 27 January 1992)

Abstract--An in vitro model was designed to study the role of ischemia/reperfusion and endothelium-derived oxygen free radicals on neutrophil adhesion, with particular interest in the endothelial adhesion moleculesinvolved. Human umbilical vein endothelial cells were submitted to 5 h hypoxia followed by various times (20 rain to 24 h) of reoxygenation. Human resting neutrophils were added to monolayers for the last 15 min ofreoxygenation. Adherence was evaluated by myeloperoxidaseassay. Under these conditions, we found an increased adhesion of neutrophils with two peaks after 20 min and 4 h reoxygenation.This was correlated with the respective expression of the preformed granule membrane protein 140 (GMP-140) and of the de novo synthesized endothelial leukocyte adhesion molecule 1 (CLAM-I) on endothelial surface. Superoxide dismutase and/or catalase, or oxypurinol added to cultures before hypoxia efficiently prevented neutrophil adhesion. These results underline the crucial role played by endothelial oxy radicals at reoxygenation in adhesion ofleukocytes, which could lead to an amplification of the oxidative stress injury. The protection offered by free radical scavengers emphasizes the potential therapeutic use of antioxidants in postischemic vascular disorders. Keywords--Hypoxia/reoxygenation,Endothelial cells, Neutrophil adhesion, Free radical scavengers,Adhesive molecules, Free radicals

INTRODUCTION

bodies, 12 is rapidly redistributed to the plasma m e m brane and also serves as a ligand. 13,14 In addition, it has been recently reported that endothelial m e m brane-associated platelet activating factor (PAF) could mediate neutrophil adhesion. 15 Although m u c h attention has been devoted to the importance of the enhanced neutrophil adhesion occurring in microvessels during postischemic periods, 3-7 the mechanisms responsible for such adherence have not been studied extensively yet. By use of various free radical scavengers, recent studies have underlined the involvement of superoxide anion and hydrogen peroxide in this p h e n o m e n o n . 16,17 However, the role of oxy radicals largely produced by endothelial cells at reperfusion 18-22 remains to be defined more clearly. The purpose of the present study was therefore to investigate the relationship between hypoxia/reoxygenation (H/R)-induced endothelial free-radical generation and adhesion of resting neutrophils, with particular interest in the adhesion molecules involved.

Localized adhesion of circulating neutrophils to endothelial lining is a critical step in a variety ofpathophysiological processes, including inflammation 1,2and ischemia/reperfusion. 3-7 This complex cell/cell interaction requires an increased expression of surface adhesive molecules either on neutrophils or on endothelial cells. On the one hand, it is well known that chemoattractant (leukotriene B4, C5a, etc.)-activated neutrophils express more C D 1 1 / C D 1 8 molecules. 2,8 On the other hand, cytokine (Interleukin-l, t u m o r necrosis factor)-activated endothelial cells express two adhesive proteins, namely endothelial leukocyte adhesion molecule 1 (CLAM- 1)9'1° and intercellular adhesion molecule 1 (ICAM- I ). ~0,~1U p o n various stimuli, including thrombin, histamine, and hydrogen or lipid peroxides, the preformed granule m e m b r a n e protein 140 (GMP-140), located in Weibel-Palade

Address correspondence to: Guy Modat. 21

22

(). I)ALLUY ~'I ~I/ MATERIALS AND M E T H O D S

Cell culture Human umbilical vein endothelial cells (HUVEC) were isolated from fresh cords by treatment with 0.1% collagenase in Dulbecco's modified Eagle's medium (DMEM). Primary isolates were cultured and further subcultured in 0.2% gelatin-coated dishes in M199 Earle medium supplemented with 20% heat-inactivated fetal calf serum (FCS), L-glutamine (4 raM), penicillin (100 U/mL), streptomycin (100 ug/mL), amphotericin (2.5 tag/mL), and heparin (10 U/mE). Endothelial cell growth supplement (300 ug/mL) was added to cultures every 2 d. The endothelial origin of monolayers was verified by phase contrast microscopic cobblestone appearance at confluence and the presence of factor VIII related antigen. 23

Isolation ~?fineutrophils Human neutrophils from healthy adult volunteers were isolated from heparinized venous blood by dextran sedimentation followed by centrifugation on Ficoll-hypaque as previously described. 24 Contaminating erythrocytes were removed by hypotonic lysis in 0.75% NH4C1 solution. Neutrophils were resuspended in DMEM supplemented with HEPES (40 mM) pH 7.4 at a final concentration of 106 cells/mL. The purity of the preparation was more than 95~, as judged after May Grunwald-Giemsa staining. Cell viability was more than 98%, as judged by trypan blue exclusion.

Hypoxia/reoxy,¢enation (H/R) treatment Tightly confluent HUVEC in 24 multiwell plates (passage level 3-4) were washed twice and covered with a minimum volume of DMEM (300 #L/well). Monolayers were exposed to 5 h hypoxia in a humidified glass chamber at 37°C in a 95% N2/5% C O 2 atmosphere. Measurements of the pO2 in supernatants (I.L. 1306 blood gas analyser, Delhomme Co., Paris, France) showed that hypoxia was obtained within 10 min, reaching a p O 2 plateau of 30 mm Hg. Reoxygenation was further obtained by replacing cells in normoxic conditions (95% air/5% CO2) for increasing lengths of time (20 rain to 24 h). When reoxygenation was for 24 h, the medium was replaced at the end of hypoxia by DMEM supplemented with 0.1% FCS. This procedure maintained cell survival without affecting the adhesion assay described below. When used, drugs were added to cultures before hypoxia. Controls consisted of HUVEC incubated for the same duration of various experiments in DMEM and in

normoxic conditions, with or without the diflerenl drugs tested.

Xanthine oxidase (XO) and .vanthine deto,droL,enaxc' (XD) activities HUVEC monolayers were washed with PBS and covered by an ice-cold extraction phosphate buffer (50 raM) containing triton X-100 (0.2% v/v), ethylenediaminetetraacetic acid (EDTA) (1 raM), dithiotreitol (10 raM), and phenyl-methyl sulfonyl fluoride ( 1 mM). After 5 rain incubation, the buffer was repeatedly pipeted back and forth to ensure cell lysis and extraction. XD and XO activities in cell lysate were immediately assayed by monitoring the appearance of uric acid at 293 nm in the presence (XD + XO) or absence (XO) o f N A D + (50 uM) in a phosphate buffer (50 raM) containing EDTA ( 1 raM) and xanthine (60 aM), as previously described. ~5 The enzymatic activity was expressed as mU XO or XD/mg protein. A unit was defined as the amount of enzyme inducing the formation of 1 #tool uric acid per minute.

,4 dhesion assay Whatever the experiment, adhesion assay was carried out by adding resting neutrophils (5 l05 cells/ well) to twice-washed HUVEC monolayers for the last 15 rain reoxygenation (H/R treatment) or normoxia (control). Nonadherent neutrophils were removed by two washes, and the content of each well was solubilized with 0.5% hexadecyltrimethyl ammonium bromide in phosphate buffer (50 mM) adjusted to pH 6. Neutrophil adhesion was evaluated by the measure of myeloperoxidase activity in the extracts. In some experiments, HUVEC were fixed at the end ofreoxygenation for 10 rain with 2.5% glutaraldehyde prior to adhesion assay. In studies with the PAF antagonist BN 50730, neutrophils were preincubated tbr 10 min with the drug (0.1 raM) before use. Microscopic observation of monolayers, before and after neutrophil adhesion, showed no detachment or retraction of endothelial cells leading to exposure of extracellular matrix.

Molecule adhesion antibodies Three monoclonal blocking antibodies directed against GMP-140, ELAM-1, and ICAM-I, respectively, IOP62 (5 ug/mL), BBA2 (50 ug/mL), and F2B 1.8 (1 ug/mL), were used. They were added to HUVEC monolayers 1 h before adhesion assay. Neutrophils were preincubated with heat-inactivated human

Hypoxia/reoxygenation and neutrophil adhesion

gamma globulins (10 mg/mL) for 30 min at 4°C and resuspended in DMEM/HEPES before use.

Myeloperoxidase (MPO) assay MPO was assayed by slight modifications of a previously reported spectrophotometric method, z6 Briefly, 50 ~L aliquots of well extracts were mixed with 700 uL phosphate buffer, pH 6, containing 0.167 mg/mL O-dianisidine dihydrochloride and 5 10-4% hydrogen peroxide. Absorbance change was measured at 460 nm over 3 min. MPO variation was verified to be linearly and similarly correlated with neutrophil concentrations in the presence or absence of HUVEC, whatever the treatments.

Von Willebrand factor (vWF) measurement vWF content of culture supernatants was evaluated by enzyme linked immunosorbent assay (ELISA). A standard curve was constructed by using dilutions of normal human plasma for vWF quantitation in samples studied, in triplicate. One milliliter of normal plasma is defined to contain 1 U (10 ug) of vWF.

Reagents Catalase (from bovine liver), cycloheximide, dithiothreitol, endothelial cell growth supplement, hexadecyltrimethyl ammonium bromide, human gamma globulins, hydrogen peroxide, mepacrine, O-dianisidine dihydrochloride, oxypurinol, pentamidine, phenyl-methyl sulfonyl fluoride, and superoxide dismutase (from bovine erythrocyte) were purchased from Sigma Chemical Co. (St. Louis, MO). Dextran T 500 and Ficoll Hypaque were from Pharmacia (Uppsala, Sweden). Triton X-100 was from Merck (Darmstadt, Germany). BN 50730 was kindly provided by Dr. P. Braquet (Institut Beaufour, Paris, France). mAb B B A 2 was from British Bio-technology (Oxon, UK). mAb IOP62 was from Immunotech (Marseille, France). mAb F2B 1.8 was given by Dr. P. Carayon (Sanofi, Montpellier, France). Antihuman IL-la and IL-1/3 Abs were obtained through the courtesy of Dr. J. Dornand (Inserm U 65, Montpellier, France). Assechrom vWF ® was purchased from Stago (Asnirres, France). Tissue culture materials were from ATGC (Paris, France). All other chemicals used were of the highest grade available.

23

Statistical analysis All results are expressed as mean + SEM. Statistical differences between means were determined using analysis of variance followed by Student t test, Dunnett's test, or Newman-Keuls's test according to experiments. Results were considered significant at P < .05. RESULTS

Time course of resting neutrophil adhesion to H/Rtreated endothelial cells When confluent HUVEC were exposed to 5 h hypoxia and reoxygenated, a time-related increase in neutrophil adherence to monolayers was observed as compared to corresponding normoxic controls (Fig. 1). A first peak was found after only 20 min reoxygenation. It was very transient, since adhesion significantly decreased 10 min later. A more pronounced adhesion was further observed after 4 h reoxygenation. Return to basal adhesion occurred after 24 h reoxygenation. Figure 1 also shows that HUVEC immediately fixed in glutaraldehyde after hypoxia (time 0 reoxygenation) bound neutrophils as fixed controls. This demonstrated that hypoxia alone had no effect on neutrophil adhesion. As clearly indicated in Table 1, the fixation procedure used did not modify the adhesion assay, since no significant difference was found between fixed and unfixed HUVEC after 20 min and 4 h reoxygenation. Moreover, these experiments avoided any effect of a potential chemoattractant release from endothelial cells2v and subsequent neutrophil activation for the duration of the adhesion assay. This was further confirmed by experiments using fixed neutrophils (data not shown).

Effect ~f free radical scavengers on H/R-induced neutrophil adhesion Regarding the kinetics of adherence in our model, studies have been focused on the maxima observed (i.e., at 20 min and 4 h reoxygenation following 5 h hypoxia). The addition of superoxide dismutase (30 and 300 U/mL) to HUVEC monolayers before hypoxia prevented neutrophil adhesion in a dose-dependent manner. The highest protection (more than 75%) was found for 300 U/mL on the 4 h reoxygenation peak of adhesion (Fig. 2). Catalase (30 and 300 U/mL) similarly prevented adhesion. In addition, we found that a combination of both enzymes (30 U/mL each) produced a synergistic effect, again more pronounced after 4 h reoxygenation. Heat-inactivated enzymes were ineffective, indicating that specific en-

24

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Fig. I. Kinetics of neutrophil adhesion to H/R-treated H U V E C monolayers. Adherence was evaluated by myeloperoxidase measurements. A rate of absorbance of 0.012/min corresponded to l04 neutrophils. T i m e 0 reoxygenation was obtained by using glutaraldehyde fixed HUVEC. Data are m e a n s _+SEM of 6 to 10 experiments, each in triplicate. *p < .05: **p < .01 relative to corresponding controls.

zymatic activities were required for the protective effect. The involvement of superoxide anion in the phenomenon was further confirmed by the detection of an increased XO activity in HUVEC after 20 min and 4 h reoxygenation, as compared to control cells, which mainly possessed XD activity (Table 2). Moreover, as shown in Figure 2, the xanthine oxidase inhibitor oxypurinol (0.2 mM) was able to prevent neutrophil adhesion with the same efficacy whatever the duration of reoxygenation. Figure 2 also shows that mannitol (1 mM) and ethanol (20 mM), two known hydroxyl radical scavengers, z8 significantly prevented adherence after only 4 h reoxygenation. Endothelial adhesion molecules involved Among molecules expressed by activated endothelial cells to bind neutrophils, some including GMP-

14013 and PAF 14act rapidly and transiently after stimulation. In contrast, those requiring de novo protein synthesis, such as ELAM-I and ICAM-I, 1° appear

Table 1. Influence of H U V E C Fixation on H/R-induced Neutrophil Adherence ~,~ Increase in Adhesion Length of Reoxygenation 20 min 4h

Unfixed H U V E C

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H U V E C were fixed before the adhesion assay as indicated in Materials and Methods. Results are expressed as % increase in adhesion from corresponding fixed and unfixed controls ( H U V E C in normoxic conditions for the duration of experiments). Data are m e a n s _+ SEM of 5 to 10 experiments each in triplicate. " No significant difference was found between the two groups of fixed and unfixed HUVEC.

Hypoxia/reoxygenation and neutrophil adhesion

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