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(Circulation Research. 1997;81:922-931.)
© 1997 American Heart Association, Inc.

Articles

Molecular Mechanisms of Anoxia/Reoxygenation-Induced Neutrophil Adherence to Cultured Endothelial Cells

Hiroshi Ichikawa, Sonia Flores, Peter R. Kvietys, Robert E. Wolf, Toshikazu Yoshikawa, D. Neil Granger, , Tak Yee Aw

From the Departments of Physiology and Medicine (H.I., S.F., D.N.G., T.Y.A.) and the Center of Excellence in Arthritis and Rheumatism (R.E.W.), LSU Medical Center, Shreveport, La; the A.C. Burton Vascular Research Laboratory (P.R.K.), Victoria Hospital Research Institute, London, Ontario, Canada; and the First Department of Internal Medicine (T.Y.), Kyoto (Japan) Prefectural University of Medicine.

Correspondence to Dr Tak Yee Aw, Department of Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. E-mail taw{at}lsumc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract The objectives of this study were to (1) determine the time course of neutrophil adhesion to monolayers of human umbilical vein endothelial cells (HUVECs) that were exposed to 60 minutes of anoxia followed by 30 to 600 minutes of reoxygenation and (2) define the mechanisms responsible for both the early (minutes) and late (hours) hyperadhesivity of postanoxic HUVECs to human neutrophils. The results clearly demonstrate that anoxia/reoxygenation (A/R) leads to a biphasic increase in neutrophil adhesion to HUVECs, with peak responses occurring at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Oxypurinol and catalase inhibited phase-1 adhesion, suggesting a role for xanthine oxidase and H₂O₂. In comparison, platelet activating factor (PAF) contributed to both phases of neutrophil adhesion. Anti–intercellular adhesion molecule-1 (ICAM-1) and anti–P-selectin antibodies (monoclonal antibodies [mAbs]) attenuated phase-1 neutrophil adhesion, consistent with roles for constitutively expressed ICAM-1 and enhanced surface expression of preformed P-selectin. Phase-2 neutrophil adhesion was attenuated by an anti–E-selectin mAb, indicating a dominant role of this adhesion molecule in the late phase response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides containing the nuclear factor-B or activator protein-1 cognate DNA sequences significantly attenuated phase-2 response, suggesting a role for de novo macromolecule synthesis. Surface expression of ICAM-1, P-selectin, and E-selectin on HUVECs correlated with the phase-1 and -2 neutrophil adhesion responses. Collectively, these findings indicate that A/R elicits a two-phase neutrophil–endothelial cell adhesion response that involves transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules.

Key Words: nuclear transcription factor • endothelial cell adhesion molecule • leukocyte–endothelial cell adhesion • selectin • intercellular adhesion molecule-1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well recognized that the microvasculature is highly sensitive to I/R and that hyperadhesiveness of leukocytes to endothelial cells contributes to I/R-induced tissue injury.¹ In an effort to define the mechanisms responsible for reperfusion-induced vascular injury, a number of in vitro models have been developed to simulate the responses of endothelial cells to I/R. Because of its simplicity, many investigators have used monolayers of cultured endothelial cells exposed to A/R as a model system to mimic I/R-induced vascular changes in vivo.^2–4 These in vitro approaches yield responses that mimic changes in the microvasculature that often accompany I/R in vivo, ie, leukocyte–endothelial cell adhesion,^5–8 increased endothelial monolayer permeability,^9,10 and increased oxidant stress.^11,12

Although the various in vitro models of I/R-induced NECA have largely yielded consistent results, there are some significant discrepancies between studies that appear to relate to differences in the magnitude and duration of the hypoxic insult as well as the times at which neutrophil adhesion was monitored after reoxygenation. For instance, different peak responses of leukocyte–endothelial cell adhesion have been reported, depending on the duration of anoxia and/or reoxygenation. We previously reported significant increases in neutrophil adherence to endothelial cells at 30 minutes of reoxygenation following 30 minutes of anoxia,^1,2 which is consistent with an early and rapid inflammatory response to A/R. Other investigators have found that exposure of endothelial cell monolayers to hypoxia (PO₂, 30 mm Hg) for 5 hours followed by reoxygenation for up to 24 hours exhibited not only a transient peak of neutrophil adhesion at 20 to 30 minutes but also a second adhesion response at 4 hours after reoxygenation.³ Discrepant findings also exist regarding the contribution of different adhesion molecules to A/R-induced NECA. For example, although E-selectin antibodies do not attenuate A/R-induced NECA at early periods (30 minutes), some investigators have demonstrated an inhibitory effect of these antibodies when neutrophil adhesion is examined at longer periods after reoxygenation.³ Collectively, these different findings, which are based on data obtained at various times after reoxygenation, suggest that A/R elicits time-dependent multiphase leukocyte–endothelial cell interactions that are likely to be mediated by different mechanisms.

At present, relatively little is known about the intracellular mediators and molecular determinants of the early and late phases of the NECA that is elicited by A/R. Current evidence based on studies with antioxidants implicates a role for oxidants such as H₂O₂ in A/R-mediated leukocyte hyperadhesiveness to endothelial cells.^8,13 Several studies have used H₂O₂ as a standard oxidizing agent to evaluate neutrophil–endothelial cell interactions, endothelial monolayer permeability, expression of adhesion molecules, and activation of nuclear transcription factors^14,15 in response to cellular oxidative stress. These studies suggest that H₂O₂ mimics many of the inflammatory and vascular responses elicited by I/R.^5,8,13

The objectives of the present study were to determine the time course of the NECA responses elicited over several hours after reoxygenation of anoxic endothelial cell monolayers and to define the mechanisms responsible for both the early (minutes) and later (hours) NECA responses induced by A/R. Another objective was to determine whether the two-phase NECA response caused by A/R can be mimicked by physiologically relevant concentrations of H₂O₂.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
The following chemicals were obtained from Sigma Chemical Co: thymidine, Histopaque 1077, SOD, catalase, oxypurinol, and 3-aminobenzamide. Endothelial cell growth medium was purchased from Clonetics. Endothelial cell growth factor and Dil-Ac-LDL were from Biomedical Technologies, Inc. Fetal calf serum was obtained from Hyclone Laboratories Inc. Mouse anti-human factor VIII was from Calbiochem. The mAbs used in the present study were TS1/18 (murine IgG1 anti-human CD18),¹⁶ RR1/1 (murine IgG1 anti-human ICAM-1),^17–19 PB1.3 (murine IgG1 anti-human P-selectin),^20,21 and CL3 [murine IgG1 F(ab')₂ anti-human E-selectin].²² RR1/1 was a gift from Dr Robert Rothlein at Boeringer-Ingelheim (Ridgefield, Colo). TS1/18, PB1.3, and CL3 were provided by Dr Donald Anderson from Pharmacia-UpJohn Laboratories (Kalamazoo, Mich). The proteasome inhibitor, MG132, was a gift from Dr Steve Brand from ProScript, Inc (Cambridge, Mass). The antioxidants, PDTC and H15, were gifts from Dr Russell Medford from Emory University (Atlanta, Ga). The PAF receptor antagonist, WEB 2086, was obtained from Boehringer-Ingelheim (Ingelheim, FRG). The sICAM-1 ELISA kit was purchased from Cayman Chemical Co.

Subjects
The procedures used to obtain human neutrophils and human umbilical cords were approved by the Institutional Review Board for Human Research at the Louisiana State University Medical Center. Each subject donating blood provided written consent and was compensated for participating in the study. Freshly discarded human umbilical cords were obtained from the delivery suite of Louisiana State University Medical Center.

Cell Culture and Treatment Protocols
HUVECs were harvested from umbilical cords by collagenase treatment as previously described.² The cells were grown in endothelial cell growth medium supplemented with 10% heat-inactivated fetal calf serum, thymidine (2.4 mg/L), glutamine (230 mg/L), heparin sodium (10 IU/mL), antibiotics (100 IU/mL penicillin, 100 mg/mL streptomycin, and 0.125 mg amphotericin B), and endothelial cell growth factor (10 ng/mL). The cell cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂ and expanded by brief trypsinization with 0.25% trypsin in phosphate-buffered saline containing 0.02% EDTA. Primary passage HUVECs were seeded onto 11-mm 48-well tissue culture plates coated with gelatin (0.1%) and fibronectin (25 µg/mL). Culture medium was replaced every second day. Passage-1 cultures were used for the studies. Cells were identified as endothelial cells by their cobblestone appearance at confluence and positive labeling with acetylated LDL labeled with Dil-Ac-LDL or mouse anti-human factor VIII.

Neutrophils
Human neutrophilic PMNs were isolated from venous blood of healthy adults using standard dextran sedimentation and gradient separation on Histopaque 1077.^2,4 This procedure yields a PMN population that is 95% to 98% viable (by trypan blue exclusion) and 98% pure (by acetic acid–crystal violet staining).

A/R Protocol
The in vitro model of A/R used in the present study is similar to that previously reported.² Briefly, confluent HUVEC monolayers were exposed to anoxia by incubating in a Plexiglas chamber that was continuously purged (1 L/min) with an anoxic gas mixture (93% N₂/5% CO₂/2% H₂). To ensure an oxygen-free environment, the gas mixture was passed through a catalytic deoxygenizer (Fisher Chemical) before entry into the chamber. Chamber PO₂ was monitored during the entire experiment using an oxygen electrode (model OM-1, Microelectrodes). Temperature in the chamber was maintained at 37°C by a heating pad. After a 60-minute period of anoxia, reoxygenation was initiated by exposing the endothelial cells to room air for periods ranging between 15 and 600 minutes. Control endothelial cells were exposed to normoxia (21% O₂/5% CO₂/74% N₂) for the duration of the experiment (normoxic controls).

Adhesion Assays
Isolated neutrophils were suspended in PBS (2x10⁷ cells/mL) and radiolabeled with 30 µCi Na⁵¹CrO₄/mL neutrophil suspension at 37°C for 1 hour. The cells were washed twice with 4°C PBS, spun at 250g for 8 minutes to remove unincorporated radioactivity, and resuspended in plasma-free HBSS. Labeled neutrophils were added to HUVEC monolayers at a neutrophil-to-HUVEC ratio of 10:1. After coincubation (30 minutes), the percentage of added neutrophils that adhered to the HUVEC monolayers was quantified.²

To assess whether superoxide, H₂O₂, xanthine oxidase, or PAF plays a role in the A/R-induced hyperadhesivity, we treated monolayers with SOD (1000 U/mL), catalase (1000 U/mL), oxypurinol (xanthine oxidase inhibitor, 100 µmol/L), or a PAF receptor antagonist (WEB 2086, 10 µmol/L).¹⁸ SOD, catalase, or oxypurinol was added to HUVECs before exposure to A/R, whereas the PAF receptor antagonist was added to the A/R-conditioned HUVECs 30 minutes before the adhesion assay. The role of adhesion molecules was tested using the respective mAbs for CD18 (TS1/18), P-selectin (PB1.3), ICAM-1 (RR1/1), and E-selectin (CL3). All mAbs were added to the A/R-conditioned HUVECs 30 minutes before the adhesion assay. To determine whether transcription and/or translation is involved in the A/R-dependent increase in PMN adherence, HUVECs were exposed to either ActD (2 µg/mL) or CHX (1 µg/mL) before A/R. The contribution of the nuclear transcription factors, AP-1 and NFB to neutrophil adherence in A/R-conditioned endothelial cells was assessed using monolayers treated with 3-aminobenzamide (AP-1 inhibitor, 1 mmol/L)^23,24 or MG132 (proteasome inhibitor, 40 µmol/L).²⁵ Both inhibitors were added to HUVEC monolayers before A/R. To further define the role of NFB and AP-1 in A/R-induced PMN adherence, HUVEC monolayers were exposed to double-stranded phosphorothioate oligonucleotides as decoys for the respective transcription factors. The oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis²⁶ according to the manufacturer's protocol (Eppendorf). The sequence of the sense strand of the B oligonucleotide (B-PT) was 5'-AGGGACTTTCCGCTGGG GACTTTCC-3',²⁷ and that of the AP-1 oligonucleotide was 5'-CGCTTGATGAGTCAGCCGGAA-3'²⁸; these sequences were annealed to their respective antisense complementary strands. Parallel experiments were performed using the nonprotein binding B-PT sequence, 5'-AAAAGTCCCTTGCT GAAAGTCCCTT-3', or the nonprotein binding AP-1–PT sequence, 5'-CGCTTGACAGACTGGCCGGAA-3', annealed to their respective complements. HUVEC monolayers were pretreated with 20 µmol/L ds-oligonucleotides for 3 hours before anoxia. To determine whether activation of NFB was mediated by oxidants, we used the antioxidant PDTC and its analogue, H15. Both antioxidants were added to HUVEC monolayers 60 minutes before anoxia.

In a separate series of experiments, HUVEC monolayers were treated with different concentrations of H₂O₂ (50, 100, 500, and 1000 µmol/L) under normoxic conditions for 15 to 600 minutes. ⁵¹Cr-labeled neutrophils were added to the endothelial cell monolayers after oxidant exposure, and neutrophil adherence was determined 30 minutes later.

sICAM-1 Assay
To determine the levels of sICAM-1, media from A/R-conditioned HUVEC monolayers at phase 1 and phase 2 were obtained, and the adhesion molecule was quantified using a commercially available ELISA kit as previously described.^29,30

ELISA of ECAM Expression
HUVECs were plated on 48-well tissue culture dishes. Primary antibodies for either ICAM-1, P-selectin, or E-selectin in HBSS/PBS with 5% FBS were added to each well and incubated for 30 minutes at 37°C. The cells were washed and incubated with the secondary antibody, horseradish peroxide–conjugated goat anti-mouse IgG (IgG1+IgG2a+IgG2b+IgG3, Southern Biotechnology Associates, Inc) diluted 1:500 in HBSS/PBS with 5% FBS for 30 minutes. The wells were then washed, and the binding of antibody was detected by the addition of 100 µL of 0.1 mg/mL 3,3',5,5'-tetramethylbenzidine (Sigma Chemical Co) with 0.003% H₂O₂. The reaction was stopped by the addition of 25 µL of 8N sulfuric acid. The samples were transferred to 96-well plates, and color development was read on a spectrometer (Titertek Multiskan MCC/340, ICN) at an optical density of 450 nm after subtracting the background values in cells stained only with the second-step antibody. All data points were performed at least in duplicate.

Preparation of Nuclear Extracts
HUVECs were plated on P-100 tissue culture dishes. Confluent HUVECs were exposed to anoxia for 60 minutes and then reoxygenated for 240 minutes. Nuclear extracts were prepared by a modification of the method of Dignam et al.³¹ Briefly, after washing with PBS, cells were centrifuged, and the cell pellet was suspended in 500 µL of hypotonic buffer (10 mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl₂, 10 mmol/L KCl, and 0.5 mmol/L DTT). After recentrifugation, the cells were resuspended in 1 mL of hypotonic buffer containing 0.1% IGEPAL (Sigma Chemical Co) by gentle homogenization to disrupt the cell membrane. After standing for 5 minutes at 4°C, the homogenate was centrifuged, and the nuclear pellet was resuspended in 100 µL of low salt buffer (20 mmol/L HEPES, pH 7.9, 0.2 mmol/L EDTA, 25% [vol/vol] glycerol, 1.5 mmol/L MgCl₂, 20 mmol/L KCl, and 0.5 mmol/L DTT). Thereafter, 100 µL of hypertonic buffer (10 mmol/L HEPES, pH 7.9, 0.1 mmol/L EDTA, 50 mmol/L KCl, 300 mmol/L NaCl, 10% [vol/vol] glycerol, and 0.5 mmol/L DTT) was added in a dropwise manner. This suspension was incubated for 30 minutes at 4°C, followed by centrifugation at 17 000g for 5 minutes. The resulting supernatant (nuclear extract) was stored at -70°C. Protein concentrations were determined by the Bradford³² method. To minimize proteolysis, all buffers contained 0.2 mmol/L phenylmethylsulfonyl fluoride.

EMSAs
A [³²P]-radiolabeled ds-DNA probe for NFB and AP-1 was generated from [-³²P]ATP and ds-oligonucleotides in a kinase reaction. The B and AP-1 ds-oligonucleotides used for the EMSAs were the same as those used as inhibitors in the adhesion molecule expression assay. The oligonucleotides (3.5 pmol) were 5' end–labeled with T4 polynucleotide kinase and [-³²P]ATP (10 µCi), according to manufacturer's specifications (Promega) at 37°C for 30 minutes, in a buffer containing 70 mmol/L Tris-HCl (pH 7.6), 10 mmol/L MgCl₂, and 5 mmol/L DTT. The reaction was stopped by the addition of EDTA to 50 mmol/L, and the volume was adjusted to 100 µL with TE buffer (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA). A typical binding reaction consisted of 10 µg of HUVEC nuclear proteins, 1x10⁵ cpm of the synthetic ³²P-labeled oligonucleotides, 4% glycerol, 1 mmol/L MgCl₂, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), and 0.5 mg/mL poly(dI-dC) with or without 50-fold molar excess of unlabeled competitor in a total volume of 20 µL. After incubation at room temperature for 30 minutes, the complexes were resolved on a 4% nondenaturing polyacrylamide (monomer-to-bis ratio of 40:1) gels at 35 mA for 3 hours at room temperature in 0.5x Tris–boric acid EDTA buffer.³³ The gels were dried and autoradiographed. Band densities were determined by computer-assisted image analysis of autoradiograms using an NIH Image 1.57 software program. Intensity of each band was corrected for the respective background. For supershift assays, antibodies against p50 or p65 (Santa Crutz Biotechnology) were included in the binding reactions.

Statistical Analysis
All values are expressed as mean±SE. Data were analyzed using a one-way ANOVA with Bonferroni corrections for multiple comparisons.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fig 1 illustrates the time course of A/R-induced neutrophil adherence to HUVEC monolayers. The data show that 60 minutes of anoxia followed by reoxygenation resulted in enhanced adhesivity of neutrophils to HUVECs, with peak adhesion responses at 30 minutes (phase 1) and 240 minutes (phase 2), indicating that A/R elicits early- and late-phase inflammatory responses. The A/R-induced hyperadhesivity of neutrophils to HUVECs in phase 1 is similar to results previously published from our laboratory.² Light microscopy of HUVEC monolayers with or without neutrophils shows that significant adhesion of neutrophils to endothelial cells occurred at 30 and 240 minutes of reoxygenation after anoxia but not at the corresponding times after normoxia (data not shown). Moreover, exposure of HUVEC monolayers to 60 minutes of anoxia followed by 30 minutes and 240 minutes of reoxygenation with or without neutrophils did not disrupt the monolayers, and neutrophils were not binding to the matrix. The postanoxic HUVEC monolayers were similar in appearance to the those exposed to normoxia for the same experimental period. Furthermore, we observed no transmigration of adherent neutrophils through the endothelial cell monolayers under all experimental conditions. Taken together, these results suggest that the phase-1 and phase-2 neutrophil adhesions to endothelial cells induced by A/R are specific.

View larger version (19K): [in this window] [in a new window]   Figure 1. Time-course of neutrophil adherence to HUVECs after A/R. HUVEC monolayers were exposed to 60 minutes of anoxia (or normoxia) and then reoxygenated. 51Cr-labeled neutrophils were added to HUVEC monolayers on reoxygenation, and neutrophil adherence was determined 30 minutes later. Each value represents mean±SE of four to six experiments performed in triplicate.

Fig 2 shows the effect of catalase, SOD, oxypurinol, and a PAF receptor antagonist (WEB 2086) on A/R-induced NECA during phase 1 and phase 2. The increased adherence to A/R-conditioned HUVECs was significantly diminished by catalase and oxypurinol at phase 1, but not phase 2. However, the PAF receptor antagonist significantly inhibited A/R-induced leukocyte adherence at both phases. SOD was without effects at both phases.

View larger version (42K): [in this window] [in a new window]   Figure 2. Effect of catalase (CAT), SOD, oxypurinol (OXY), and PAF receptor antagonist (WEB 2086) on A/R-induced neutrophil adhesion to HUVECs. Responses are shown for neutrophil adhesion at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Each value represents mean±SE of four to six experiments performed in triplicate. *P<.05 compared with normoxic controls; #P<.05 and ##P<.01 compared with untreated controls (A/R only).

Fig 3 summarizes the effects of mAbs directed against ECAMs on neutrophil adherence to endothelial monolayers subjected to A/R at phase 1 and phase 2. The increase in neutrophil adherence to HUVECs was significantly reduced, in both phases, by anti–P-selectin (PB 1.3). Anti–ICAM-1 (RR1/1) also significantly reduced neutrophil adherence to A/R-conditioned HUVECs in phase 1. Interestingly, the standard saturating anti–ICAM-1 dose of 20 µg/mL, which effectively blocks the phase-1 adhesion response, had no effect on neutrophil adhesion at phase 2. The anti–E-selectin–specific antibody (CL3) significantly inhibited leukocyte adhesion to HUVECs in phase 2, but had no effect in phase 1. When anti–P-selectin and –E-selectin were used in combination, the increased NECA was further attenuated compared with either mAb alone (data not shown). The CD18-specific mAb largely abolished the leukocyte adhesion to HUVECs exposed to A/R in both phases (data not shown).

View larger version (44K): [in this window] [in a new window]   Figure 3. Effect of mAbs to P-selectin (PB1.3, 20 µg/mL), ICAM-1 (RR1/1, 20 µg/mL), and E-selectin (CL3, 20 µg/mL) on neutrophil adherence to HUVECs in phase 1 and phase 2 of reoxygenation. Responses are shown for neutrophil adhesion at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Each value represents mean±SE of four to six experiments performed in triplicate. *P<.05 compared with normoxic controls; #P<.05 and ##P<.01 compared with untreated controls (results without antibody).

To determine whether the inability of the standard saturating dose of anti–ICAM-1 (20 µg/mL) to attenuate leukocyte–endothelial cell adhesion in phase 2 was due to a higher membranous ICAM-1 concentration, we exposed the monolayers to progressively increasing concentrations of the ICAM-1 mAb. The results in Fig 4 show that the enhanced neutrophil adherence to A/R-conditioned HUVECs in phase 2 was dose-dependently decreased by the anti–ICAM-1 antibody. A significant reduction in the phase-2 adhesion response was noted at twice the standard saturating mAb dose (40 µg/mL). In addition, we quantified sICAM-1 levels in the incubation medium of A/R-conditioned HUVECs in phase 1 and 2 (Fig 5). The results show that sICAM-1 levels in the incubation medium of A/R-conditioned HUVEC monolayers were significantly higher in phase 2 compared with control levels. sICAM-1 levels were unchanged in phase 1.

View larger version (58K): [in this window] [in a new window]   Figure 4. Effect of increasing concentration of a mAb to ICAM-1 (RR1/1) on neutrophil adherence to HUVECs at 240 minutes of reoxygenation (phase 2). Each value represents mean±SE of four to six experiments performed in triplicate. *P<.05 and **P<.01 compared with nontreated controls (A/R only).

View larger version (37K): [in this window] [in a new window]   Figure 5. Change in sICAM-1 levels in incubation medium of HUVECs exposed to A/R. Responses are shown for sICAM-1 at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Each value represents mean±SE of nine experiments performed in duplicate. *P<.05 compared with normoxic controls.

The possibility that H₂O₂ may elicit a biphasic NECA response similar to A/R was examined in HUVEC monolayers exposed to different concentrations of the oxidant. In these experiments, neutrophil adhesion was monitored at 30 minutes (phase 1) and 240 minutes (phase 2) after H₂O₂ exposure. The results show that H₂O₂ dose-dependently increased leukocyte adhesion at phase 1 and 2, with lower concentrations exerting a more profound effect on phase-2 adhesion (Fig 6). The peroxide-induced neutrophil adhesion to endothelial cells was completely abolished in phase 1 and significantly attenuated in phase 2 by a PAF receptor antagonist (Fig 7), indicating that PAF production by endothelial cells contributes to both phases of the oxidant-mediated adhesion response. The anti–P-selectin mAb significantly reduced the hyperadhesiveness of peroxide-treated HUVECs to neutrophils in the early phase. However, in the late phase, the anti–P-selectin mAb did not inhibit the increase of adhesion levels induced by H₂O₂ (data not shown).

View larger version (62K): [in this window] [in a new window]   Figure 6. Effect of increasing concentration of H2O2 on neutrophil adherence to naive HUVECs. HUVEC monolayers were exposed to H2O2 (0.1 to 1 mmol/L) for 30 minutes (phase 1) or 240 minutes (phase 2) under normoxic conditions. Each value represents mean±SE of four to six experiments performed in triplicate. *P<.05, **P<.01, and #P<.05 compared with nontreated controls.

View larger version (25K): [in this window] [in a new window]   Figure 7. Effect of catalase (CAT) and a PAF receptor antagonist (WEB 2086) on H2O2 (1 mmol/L)–induced neutrophil adherence to normoxic HUVECs at 30 minutes (phase 1) and 240 minutes (phase 2). Each value represents mean±SE of four to six experiments performed in triplicate. *P<.05 compared with normoxic controls; #P<.05 and ##P<.01 compared with untreated controls (H2O2 only).

The finding of an enhanced inflammatory response 4 hours after reoxygenation, coupled to the evidence implicating E-selectin (which is not constitutively expressed on HUVEC), suggests that A/R elicits a transcription-dependent upregulation of ECAMs. Previous studies have invoked a role for NFB and/or AP-1 in cytokine- and H₂O₂-mediated inflammatory reactions.^14,15,24,25 To assess the contribution of these nuclear transcription factors to the A/R-induced enhancement of neutrophil adhesion, HUVEC monolayers were treated with inhibitors of macromolecule synthesis (CHX, 1 µg/mL, or ActD, 2 µg/mL), a proteasome inhibitor (MG132, 40 µmol/L), or an inhibitor of AP-1 (3-aminobenzamide, 1 mmol/L). In other experiments, HUVECs were treated with ds-phosphorothioate oligonucleotides (20 µmol/L each) as decoys for B and AP-1. The results in Table 1 show that inhibition of protein synthesis significantly attenuated the A/R-induced neutrophil adherence in phase 2, but not in phase 1. Moreover, blockade of NFB and AP-1 activation significantly decreased the adhesion response in phase 2. Interestingly, the inhibitory effects of these agents on neutrophil adhesion to HUVECs in phase 2 were not complete. When anti–ICAM-1 antibody (60 µg/mL) was added to cells treated with ActD, MG132, aminobenzamide, or ds-phosphorothioate oligonucleotides, a further attenuation of the increased NECA response in phase 2 to baseline values was observed. Finally, the increased neutrophil adhesion response induced by A/R in phase 2 was also attenuated by the antioxidants, PDTC and H15 (100 µmol/L each).

View this table: [in this window] [in a new window]   Table 1. Effect of Inhibitors of Macromolecule Synthesis, NFB, and AP-1 on A/R-Induced NECA

To document that the phase-2 transcription-dependent adhesion response is associated with NFB activation, we performed EMSAs on nuclear extracts prepared from A/R-conditioned HUVECs after 240 minutes of reoxygenation. The results are shown in Fig 8. Two specific nucleoprotein adducts were evident on the gel shift (designated as upper and lower bands, Fig 8A). Supershift analyses (see Fig 9) on these retarded products suggest that the faster migrating band represents the p50 homodimer and that the slower migrating band is mostly likely the p50-p65 heterodimer. Quantification of the nucleoprotein adducts is shown in Fig 8B. The results show that reoxygenation for 240 minutes increased the amount of the slower migrating adduct (Fig 8A and 8B). Pretreatment with the B decoy almost completely abolished binding of the slower moving adduct to the radiolabeled oligonucleotide (Fig 8A, lane 5, and Fig 8B), suggesting that the inhibition by this decoy is specific. Nonbinding decoys had no effect on NFB binding (data not shown). The proteasome inhibitor, on the other hand, resulted in a significant decrease in the faster migrating adduct but an enhancement of binding of the slower migrating adduct (Fig 8, lane 4, and Fig 8B). This observation suggests that MG132 may inhibit NFB activation without affecting nuclear translocation, a suggestion that is consistent with recent findings by Cobb et al,³⁴ who showed that the proteasome inhibitor, nor-LEU, inhibits NFB activation and blocks interleukin 1ß–induced vascular cell adhesion molecule-1 and ICAM-1 gene expression in HUVECs without preventing nuclear translocation of NFB. When antibodies to p50 or p65 were added to the binding reaction before addition of the labeled oligonucleotide, we observed a supershift and a decrease in the intensity of the faster or slower migrating adducts, respectively (Fig 9, lanes 2 and 3). In these studies, we found that the binding of the p65 antibody was considerably weaker than binding of the p50 antibody. In order to visualize the supershifted products, the gel was overloaded and overexposed, which also revealed the presence of some minor adducts. These, however, were nonspecific, since we were unable to eliminate them when molar excess of unlabeled oligonucleotide was used as a nonspecific competitor (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 8. EMSAs on nuclear extracts from A/R-conditioned endothelial cells after 240 minutes reoxygenation pretreated with or without ds-oligonucleotide (20 µmol/L) or the proteasome inhibitor (MG132, 40 µmol/L). The NFB-specific adducts are indicated by bold arrows and designated as upper and lower bands. Panel A shows a representative gel shift pattern from one of six experiments. Lanes 1 to 5 are nuclear extracts of cells from normoxia (lane 1), A/R (lane 2), A/R with addition of 50x unlabeled competitor in binding reaction (lane 3), A/R treated with proteasome inhibitor (lane 4), and A/R treated with ds-oligonucleotide (lane 5). Each lane was loaded with 4.5 µg nucleoprotein. Panel B shows the quantification of the NFB adducts using computer-assisted image analyses of the autoradiogram in panel A. The intensity of each adduct was corrected for the respective background of the autoradiogram, and the result is expressed as units of NFB activity relative to normoxia (NFB activity equals 1).

View larger version (55K): [in this window] [in a new window]   Figure 9. Electrophoretic mobility supershift assay on nuclear extracts from A/R-conditioned endothelial cells after 240 minutes of reoxygenation. The figure shows a representative supershift autoradiogram. The NFB-specific adducts are indicated by bold arrows and designated as upper and lower bands. Lanes 1 to 3 are nuclear extracts of cells from A/R (lane 1), A/R with addition of p50 antibody in binding reaction (lane 2), and A/R with addition of p65 antibody in binding reaction (lane 3). Each lane was loaded with 15 µg nucleoprotein.

Fig 10a shows the time course of surface expression of P-selectin in HUVECs exposed to A/R. The data show that 60 minutes of anoxia followed by 30 minutes to 10 hours of reoxygenation resulted in increased expression of P-selectin, with peak responses at 30 minutes and between 4 and 6 hours after reoxygenation. This biphasic response is similar to the NECA response induced by A/R (see Fig 1). Fig 10b shows the kinetics of E-selectin expression in HUVECs induced by A/R. E-Selectin was not expressed in phase 1 but increased significantly in phase 2, with peak expression at 4 hours. Thereafter, E-selectin expression gradually decreased and returned to baseline values by 10 hours after reoxygenation, paralleling the kinetics of the phase-2 adhesion response induced by A/R. Fig 10c shows the time course of the surface expression of ICAM-1 in HUVECs exposed to A/R. Unlike P-selectin or E-selectin, surface expression of ICAM-1 was high in phase 1 (constitutive expression), increased in phase 2, and remained elevated for 10 hours despite decreased neutrophil adhesion (see Fig 1).

View larger version (14K): [in this window] [in a new window]   Figure 10. Time course of ECAMs (P-selectin [a], E-selectin [b], and ICAM-1 [c]) on HUVECs exposed to A/R. HUVEC monolayers were exposed to 60 minutes of anoxia and then reoxygenated. Each value represents mean±SE of four to six experiments performed in duplicate. OD450 indicates optical density at 450 nm. *P<.05 and **P<.01 compared with normoxic controls.

To evaluate whether the A/R-induced induction of ECAM (P-selectin, ICAM-1, and E-selectin) expression in phase 2 is transcription dependent, we measured the surface expression of the different ECAMs in HUVECs treated with CHX (1 µg/mL) or ActD (2 µg/mL). The contributions of different transcription factors were examined using inhibitors of NFB (MG132, 40 µmol/L) and AP-1 (3-aminobenzamide, 1 mmol/L), as well as their respective binding ds-oligonucleotides (20 µmol/L). The antioxidants PDTC and H15 were also examined. The results are summarized in Table 2. Upregulation of E-selectin induced by A/R was essentially abolished by CHX or ActD. Moreover, treatment with the proteasome inhibitor (MG132), AP-1 inhibitor (3-aminobenzamide), or antioxidants was effective in reducing the E-selectin expression induced by A/R. Interestingly, inhibition of protein synthesis and transcription resulted in only a 10% to 20% reduction in the A/R-induced expression of P-selectin or ICAM-1.

View this table: [in this window] [in a new window]   Table 2. Effect of Inhibitors of macromolecule Synthesis, NFB, and AP-1 on A/R-Induced Surface Expression of ECAMs

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has long been recognized that the microvasculature is exquisitely sensitive to the deleterious effects of ischemia and reperfusion.¹ In recent years, a variety of bioactive compounds and effector cells have been invoked as mediators of reperfusion injury, including reactive oxygen metabolites, PAF, nitric oxide, platelets, mast cells, and granulocytes.^10,35–39 In addition, a number of in vitro models have been developed to simulate the in vivo responses of vascular endothelial cells to I/R and to help define the mechanisms responsible for I/R-induced vascular injury. Previous studies using these in vitro models have successfully simulated some of the major microvascular responses that are elicited in vivo by I/R, ie, enhanced expression of ECAMs, neutrophil adhesion, and an increased endothelial monolayer permeability. The results of the present study extend these previous reports by demonstrating that exposure of HUVEC monolayers to A/R leads to a biphasic pattern of NECA, with peak responses observed at 30 minutes (phase 1) and 240 minutes (phase 2) after reoxygenation. Although the magnitude of the neutrophil adhesion responses in the two phases is similar, it appears that the contribution of ECAMs and chemical mediators to the adhesion response differs between the two phases.

The results of the present study implicate a role for xanthine oxidase, H₂O₂, and PAF in the early (minutes) neutrophil adhesion response (phase 1) elicited by A/R. These findings are consistent with earlier observations reported from our laboratory.² A role for PAF in this response is supported by the inhibitory action of the receptor antagonist WEB 2086. We found that although PAF and leukotriene B₄ are each capable of enhancing the neutrophil adhesion response to HUVECs at a concentration of 0.1 µmol/L, the PAF receptor antagonist (WEB 2086, 10 µmol/L) abolished the PAF-induced adhesion but did not affect the leukotriene B₄–induced adhesion response (data not shown). These observations suggest that endothelial cells produce PAF or PAF-like substances during A/R and that WEB 2086 is a specific antagonist to PAF receptors. The observation that catalase is as effective as the PAF antagonist in blunting the phase-1 adhesion response suggests that endothelial cell–derived H₂O₂ elicits the production of PAF.⁴⁰ Yoshida et al² have reported that both WEB 2086 and catalase can ameliorate neutrophil adhesion to naive monolayers exposed to media obtained from A/R-conditioned endothelial cells. Hence, our findings, together with published observations, are consistent with the hypothesis that A/R enhances the production and liberation of H₂O₂ (in part due to xanthine oxidase) and PAF during the early stages of reoxygenation (phase 1).

In contrast to the early (phase 1) adhesion response to A/R, the enhanced neutrophil–endothelial cell interactions observed 4 hours after reoxygenation (phase 2) do not appear to involve either xanthine oxidase or H₂O₂. Nonetheless, PAF remains as a contributor to the adhesion response in phase 2 despite the absence of peroxide generation. These findings may be explained by different sources of H₂O₂ generation in phase 1 versus phase 2. One possibility is that the major source(s) of H₂O₂ in phase 1 is cell membrane–bound xanthine oxidase on endothelial cells⁴¹ and/or neutrophilic NADPH oxidase, whereas an intracellular source (eg, mitochondria) of H₂O₂ production by endothelial cells is responsible for phase 2. In such a scenario, exogenous catalase would have access to detoxify the H₂O₂ accumulating immediately outside endothelial cells in phase 1, but the enzyme would not have access to the intracellular H₂O₂ generated in phase 2. Since PAF appears to be formed in both phases of A/R-induced adhesion, this would suggest either that PAF formation in phase 2 is independent of H₂O₂ generation or that extracellular and intracellular sources of H₂O₂ generation are equally effective in promoting the formation of PAF in HUVECs.

An interesting and potentially important observation of the present study is the difference in the relative contribution of various ECAMs to the enhanced neutrophil adhesion elicited in phase 1 and phase 2 of A/R. In both phases, it appears that activation of ß₂ integrins on neutrophils (presumably by PAF) contributes to the enhanced adhesion. In the present study, the CD18-specific mAb largely abolished leukocyte adhesion to HUVECs exposed to A/R in both phases. However, the relative contributions of ECAMs appear to differ between the two phases. In phase 1, P-selectin and ICAM-1, but not E-selectin, contribute to the neutrophil adhesion response. Our data also showed that P-selectin surface expression kinetics are biphasic, similar to the kinetics of the neutrophil adhesion response. E-Selectin was not expressed in phase 1 but increased significantly in phase 2, also paralleling the kinetics of the adhesion response. Surface expression of ICAM-1 was high in phase 1, increased in phase 2, and remained elevated for 10 hours despite decreased neutrophil adhesion. The pattern of participation of adhesion molecules in phase 1 could be explained by constitutively expressed ICAM-1,⁴² by a rapid mobilization of the preformed pool of P-selectin in endothelial cells,^43–45 and by the upregulation of ß₂ integrins by PAF or PAF-like substances that are liberated by endothelial cells.^46,47 In phase 2, however, E-selectin makes a contribution, in addition to P-selectin and ICAM-1, to the A/R-induced neutrophil adhesion. The phase-2 E-selectin–dependent response pattern appears to be consistent with a transcription-dependent upregulation of E-selectin and possibly ICAM-1 and P-selectin.^24,48,49 Several lines of evidence support the involvement of transcription-dependent upregulation of ECAMs.¹ Our data show that reagents that interfere with the activation of either NFB or AP-1 also inhibited phase-2 neutrophil adhesion and the increased surface expression of ECAMs, especially E-selectin.² Treatment of HUVECs with inhibitors of transcription and translation prevented A/R-induced phase-2 expression of ECAMs and neutrophil adhesion to HUVEC monolayers.³ Results obtained with EMSAs on nuclear extracts from A/R-conditioned cells after 240 minutes of reoxygenation show that A/R causes specific upregulation and activation of NFB.

Previous reports have implicated a role for NFB and AP-1 in the transcriptional regulation of different ECAMs after exposure of HUVECs to cytokines or oxidants.^24,48,49 The AP-1 inhibitor 3-aminobenzamide has been shown to be effective in attenuating H₂O₂-induced ICAM-1 upregulation,²³ whereas MG132, an inhibitor of NFB activation, has been shown to reduce the increased E-selectin expression elicited by different cytokines.²⁵ In the present study, we have demonstrated that both MG132 and 3-aminobenzamide significantly attenuate the A/R-induced neutrophil adhesion and E-selectin expression in phase 2. Furthermore, we have shown that ds-phosphorothioate oligonucleotides, which have multiple copies of the DNA binding site for NFB or AP-1, prevent the binding of these nuclear transcription factors to endogenous B or AP-1 enhancer-promotor elements, thereby specifically inhibiting NFB–or AP-1–dependent transcription. Concomitantly, these binding oligonucleotides also significantly inhibited the A/R-induced phase-2 E-selectin expression and neutrophil adhesion. Furthermore, the present data with EMSA have confirmed the suggestion that A/R results in NFB activation and induction. Taken together, these observations provide the first evidence that implicates both nuclear transcription factors in the transcription-dependent upregulation of adhesion molecules and enhanced neutrophil-endothelial adhesion elicited by A/R.

A notable finding in the present study is that the saturating dose of ICAM-1 antibody that effectively blocked neutrophil adhesion to HUVECs in phase 1 was incapable of inhibiting the elevated adhesion response in phase 2. However, we noted that when a higher concentration of the same antibody was used, the phase-2 adhesion response was attenuated. The latter observation suggests that the pool of ICAM-1 that is available for reaction with the antibody was much higher in phase 2. Indeed, this possibility is supported by our finding that the sICAM-1 level on the surface of the HUVEC monolayers was elevated during phase 2, but not during phase 1. This observation suggests that the HUVEC monolayers shed membranous ICAM-1 into the extracellular fluid in the late phase of reoxygenation. The significance of this is, at present, unclear.

Exposure of endothelial cell monolayers to H₂O₂ is frequently used as a model system to investigate endothelial cell responses to acute oxidant stress, such as A/R. The results of the present study support previous reports that H₂O₂ promotes leukocyte adhesion to endothelial cells.¹³ However, a novel observation in the present study is that exposure of HUVECs to H₂O₂ concentrations as low as 100 µmol/L elicits a response that is similar to the phase-2 response induced by A/R. Studies with the PAF antagonist also support the view that H₂O₂ is a reasonable model for A/R, since WEB 2086, which was effective in attenuating A/R-induced neutrophil adhesion in phases 1 and 2, was also equally effective in decreasing phase-1 and -2 adhesion responses in the peroxide model. Hence, these findings indicate that extracellular H₂O₂ effectively mimics the neutrophil adhesion responses that are elicited by A/R.

	Selected Abbreviations and Acronyms

 

ActD = actinomycin D AP-1 = activator protein 1 A/R = anoxia/reoxygenation CHX = cycloheximide Dil-Ac-LDL = 1,11-dioctadecyl-13,3,31,31,3-tetramethylindocarbocyanine perchlorate DTT = dithiothreitol ECAM = endothelial cell adhesion molecule EMSA = electrophoretic mobility gel-shift assay HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule-1 I/R = ischemia/reperfusion mAb = monoclonal antibody NECA = neutrophil–endothelial cell adhesion NFB = nuclear factor kappa-B PAF = platelet-activating factor PDTC = pyrrolidine dithiocarbamate PMN = polymorphonuclear leukocyte sICAM-1 = soluble ICAM-1 SOD = superoxide dismutase

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (P01 DK-43785, HL-48855, and DK-44510). We thank Loren P. Walsh for his assistance with the EMSA and supershift analyses.

Received March 13, 1997; accepted August 28, 1997.

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