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(Circulation Research. 1999;84:516-524.)
© 1999 American Heart Association, Inc.

Original Contributions

Molecular Mechanisms of Neutrophil-Endothelial Cell Adhesion Induced by Redox Imbalance

Satoshi Kokura, Robert E. Wolf, Toshikazu Yoshikawa, D. Neil Granger, Tak Yee Aw

From the Department of Molecular and Cellular Physiology (S.K., D.N.G., T.Y.A.), Louisiana State University Medical Center, Shreveport, La; Center of Excellence in Arthritis and Rheumatism (R.E.W.), Louisiana State University Medical Center, Shreveport, La; and First Department of Internal Medicine (T.Y.), Kyoto Prefectural University of Medicine, Kyoto, Japan.

Correspondence to Tak Yee Aw, PhD, Department of Molecular and Cellular Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. E mail TAW@lsumc.edu

	Abstract

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Abstract—Previous studies have implicated a role for intracellular thiols in the activation of nuclear factor-B and transcriptional regulation of endothelial cell adhesion molecules. This study was designed to determine whether changes in endothelial cell glutathione (GSH) or oxidized glutathione (GSSG) can alter neutrophil adhesivity and to define the molecular mechanism that underlies this GSSG/GSH-induced adhesion response. Treatment of human umbilical vein endothelial cell (HUVEC) monolayers for 6 hours with 0.2 mmol/L diamide and 1 mmol/L buthionine sulfoximine (BSO) decreased GSH levels and increased the ratio of GSSG to GSH without cell toxicity. These redox changes are similar to those observed with anoxia/reoxygenation. Diamide plus BSO–induced thiol/disulfide imbalance was associated with a biphasic increase in neutrophil adhesion to HUVECs with peak responses observed at 15 minutes (phase 1) and 240 minutes (phase 2). N-Acetylcysteine treatment attenuated neutrophil adhesion in both phases, which indicated a role for GSH in the adhesion responses. Interestingly, phase 1 adhesion was inversely correlated with GSH levels but not with the GSSG/GSH ratio, whereas phase 2 neutrophil adhesion was positively correlated with GSSG/GSH ratio but not with GSH levels. Intercellular adhesion molecule-1 and P-selectin–specific monoclonal antibodies attenuated the increased neutrophil adhesion during both phases, whereas an anti–E-selectin monoclonal antibody also attenuated the phase 2 response. Pretreatment with actinomycin D and cycloheximide or with competing ds-oligonucleotides that contained nuclear factor-B or activator protein-1 cognate DNA sequences significantly attenuated the phase 2 response, which implicated a role for de novo protein synthesis. Surface expression of intercellular adhesion molecule-1, P-selectin, and E-selectin on HUVECs correlated with the phase 1 and 2 neutrophil adhesion responses. This study demonstrates that changes in endothelial cell GSSG/GSH cause transcription-independent and transcription-dependent surface expression of different endothelial cell adhesion molecules, which leads to a 2-phase neutrophil–endothelial adhesion response.

Key Words: neutrophil • glutathione • endothelium • oxidation-reduction • anoxia • adhesion • diamide

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exposure of endothelial cells to anoxia/reoxygenation (A/R),¹ hydrogen peroxide (H₂O₂),² lipopolysaccharide (LPS),³ and cytokines, such as tumor necrosis factor (TNF)³ and interleukin-1 (IL-1),³ induce leukocyte adhesion by increasing the surface expression of various endothelial cell adhesion molecules (ECAMs). The multisubunit nuclear factor-B (NF-B) is one of the transcription factors involved in the expression of many inflammatory and immune response genes, which include the expression of E-selectin,^{4 5 6} intercellular adhesion molecule-1 (ICAM-1),⁶ and vascular cell adhesion molecule-1 (VCAM-1).^{7 8} Many inducers of NF-B activity, such as A/R, TNF, and IL-1, have been shown to cause cellular oxidative stress via stimulation of reactive oxygen species (ROS) production. These observations, coupled to the finding that exposure of cells to micromolar amounts of hydrogen peroxide can activate NF-B directly, suggest that enhanced generation of ROS may be a common signal for the activation of NF-B by a variety of inflammatory stimuli.

Cellular thiol status has been shown to modulate transcription factor activation of gene expression mediated by TNF, IL-1, LPS, or H₂O₂.^{9 10 11 12 13 14 15 16} Staal et al⁹ have shown that low thiol levels promote NF-B activation, whereas exogenous cysteine and N-acetyl-L-cysteine (NAC) was found to inhibit NF-B activity.^{14 15 16 17 18} Moreover, a decrease in glutathione (GSH) induced by inhibition of GSH biosynthesis was shown to alter the NF-B activation responses to LPS^{14 15} or TNF.^{12 19} Cytokine-induced expression of ICAM-1 has been shown to be mediated by the redox-sensitive transcription factor, activator protein-1 (AP-1).²⁰ It is apparent from the above considerations that an altered intracellular thiol and/or disulfide status may contribute to the regulation of transcription factor activation and expression of inflammatory genes during an inflammatory reaction, such as the oxidative stress associated with A/R of endothelial cells.

The contribution of altered endothelial cell GSH or oxidized glutathione (GSSG) to the cellular and molecular alterations associated with A/R-induced inflammation is poorly understood. We recently reported that exposure of human umbilical vein endothelial cells (HUVECs) to 60 minutes of anoxia followed by up to 10 hours of reoxygenation elicits a biphasic neutrophil adhesion response that is related to endothelial oxidant production and is differentially modulated by transcription-independent (30 minutes, phase 1) and transcription-dependent (4 hours, phase 2) upregulation of endothelial cell adhesion molecules (ECAMs).¹ In the phase 2 adhesion response elicited by A/R, de novo synthesis of E-selectin was mediated by NF-B and AP-1.¹ The present study was designed to determine whether an oxidant-induced imbalance in GSH/GSSG could contribute to the increased ECAM expression and enhanced neutrophil adhesion that is elicited by exposure of HUVECs to A/R. We developed a chemical model of GSH/GSSG imbalance to simulate the effects of A/R and to address 3 specific objectives: (1) to define the kinetics of neutrophil–endothelial cell adhesion that occur in response to alterations in endothelial cell GSH/GSSG, (2) to determine the molecular mechanisms that underlie the enhanced neutrophil–endothelial cell adhesion elicited by a change in GSH/GSSG, and (3) to determine whether redox activation of NF-B and AP-1 can contribute to the expression of ECAMs and increased neutrophil adhesion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
The following chemicals were obtained from Sigma Chemical Co: Histopaque 1077, diamide, buthionine sulfoximine (BSO), diethyl maleate (DEM), actinomycin D (ActD), cycloheximide (CHX), and 3-aminobenzamide (3AB). Endothelial cell growth medium and bovine brain extract were purchased from Clonetics. 1,11-Dioctadecyl-13,3,31,31,3-tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL) were from Biomedical Technologies, Inc. FBS was obtained from Atlanta Biologicals. Mouse anti–human factor VIII was from Calbiochem. The monoclonal antibodies (MAb) used in this study were 8.4A6 (murine IgG1 anti–human ICAM-1), PB1.3 (murine IgG1 anti–human P-selectin), and CL3 [murine IgG1 F(ab')₂ anti–human E-selectin]. The MAbs 8.4A6, PB13, and CL3 were generously provided by Dr Donald Anderson from Pharmacia-Upjohn Laboratories (Kalamazoo, Mich). The proteosome inhibitor, MG132, was a gift from Dr Steve Brand from ProScript, Inc (Cambridge, Mass).

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. Freshly discarded human umbilical cords were obtained from the delivery suite of Louisiana State University Medical Center. Each subject who donated blood provided written consent and was compensated for participating in the study.

Endothelial Cell Culture
HUVECs were harvested from umbilical cords by 0.25% collagenase treatment for 20 minutes at 37°C, as previously described.²¹ The cells were grown in endothelial cell growth medium (EGM), supplemented with bovine brain extract. The cell cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂ and were expanded by brief trypsinization with 0.25% trypsin in PBS that contained 0.02% EDTA. Primary-passage HUVECs were seeded into fibronectin-coated (25 µg/mL), 11-mm, 48-well tissue culture plates. Culture medium was replaced every 2 days. Cells were identified as endothelial cells by their cobblestone appearance at confluence and positive labeling with acetylated LDL lipoprotein labeled with Dil-Ac-LDL or mouse anti–human factor VIII. Passage 1 cultures were used for the studies.

Isolation of Neutrophils
Human neutrophilic polymorphonuclear cells (PMNs) were isolated from venous blood of healthy adults with standard dextran sedimentation and gradient separation on Histopaque 1077.²² 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 this study is similar to that previously reported.¹ Briefly, confluent HUVECs monolayers were exposed to anoxia through incubation 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 with an oxygen electrode (model OM-1, Microelectrodes). Temperature in the chamber was maintained at 37°C with a heating pad. After a 60-minute period of anoxia, reoxygenation was initiated by exposing the endothelial cells to room air, followed by periods of reoxygenation ranging from 0 to 4 hours.

Modulation of Cellular GSH and GSSG
Endothelial cell GSH and GSSG levels were modified with a combination of 3 chemical thiol agents. Diamide is a cell-permeant oxidant that specifically targets the thiols of GSH and free SH groups of proteins.²³ The action of diamide induces formation of disulfide bonds via thiol-diamide intermediates,²⁴ thereby promoting the formation GSSG or protein disulfide cross-link. Consequently, the redox potential of the cell is shifted in favor of a more oxidized state, which is typically reflected in an increase in the GSSG-to-GSH ratio. BSO is a potent inhibitor of -glutamyl cysteine synthetase, the rate-limiting step in GSH synthesis.²⁵ Inhibition of GSH synthesis causes a marked decrease in the total GSH pool but has minimal effect on the GSSG/GSH ratio. DEM is a substrate for glutathione S-transferase, which catalyzes the formation of GSH-DEM conjugate. The net change in redox status with DEM treatment is a decrease in total cell GSH without changes in the GSSG/GSH ratio.

Quantification of GSH and GSSG
Endothelial cell GSH and GSSG levels were determined according to the method of Reed et al.²⁶ Briefly, the assay was based on the initial formation of S-carboxymethyl derivatives of free thiols followed by the conversion of free amino groups to 2,4-dinitrophenyl derivatives. GSH and GSSG derivatives were separated by reverse-phase ion-exchange high-performance liquid chromatography.

Treatment Protocol
In our studies, 3 treatment groups were used: diamide alone, diamide plus BSO, and DEM plus BSO. To effect changes in GSH and GSSG, confluent HUVEC monolayers were treated with these agents as follow. The cell culture medium was removed, and cells were incubated with diamide (0.2 mmol/L) or DEM (0.5 mmol/L) in fresh EGM for 15 minutes. At the end of the treatment period with diamide or DEM, the medium was removed and the cells were incubated without or with BSO (1 mmol/L) in fresh EGM to prevent resynthesis of GSH for periods ranging from 0 to 360 minutes. Treated HUVEC monolayers will be referred to as redox-altered endothelial cells. Parallel controls (redox-unaltered) were performed in which endothelial cell monolayers were incubated with fresh EGM in the absence of the thiol agents for the duration of the experiment. Untreated (ie, control, redox-unaltered) and treated (ie, redox-altered) HUVEC monolayers were used to study the effect of redox imbalance on neutrophil adhesion, ECAM expression, and NF-B activation.

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 ice-cold PBS (4°C), spun at 250g for 4 minutes to remove unincorporated radioactivity, and resuspended in plasma-free HBSS. Culture media that contained the thiol agents were removed, and HBSS was added to HUVEC monolayers. Labeled neutrophils were added to control or redox-altered 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.²⁷ To examine the role of NAC on neutrophil adherence induced by redox imbalance, naive HUVEC monolayers were pretreated with 10 mmol/L NAC for 2 hours before cells were treated with diamide and BSO.

The role of adhesion molecules was tested with blocking doses of the respective monoclonal antibodies (MAbs) for P-selectin (PB1.3), ICAM-1 (8.4A6), and E-selectin (CL3). All MAbs were added to the redox-altered HUVEC monolayers immediately before the adhesion assay. To determine whether transcription and/or translation is involved in the increased PMN adherence induced by altered GSSG or GSH, HUVECs were exposed to either ActD (2 µg/mL) or CHX (1 µg/mL) 30 minutes before cells were treated with the thiol agents. The contribution of the nuclear transcription factors, AP-1 and NF-B, to GSSG/GSH-induced neutrophil adherence was assessed with HUVEC monolayers treated with 3AB (AP-1 inhibitor, 1 mmol/L)^{28 29} or MG132 (proteosome inhibitor, 5 µmol/L).³⁰ Both inhibitors were added to HUVEC monolayers 2 hours before treatment of cells with thiol agents. To further define the role of NF-B and AP-1 in GSSG- or GSH-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 electrophoresis³¹ according to the manufacturer's protocol (Eppendorf). The sequence of the sense strand of the B oligonucleotide (B-PT) was 5'-AGGGACTTTCCGCTGGGGACTTTCC-3',³² and that of the AP-1 oligonucleotide was 5'-CGCTTGATGAGTCAGCCGGAA-3'³³ ; these sequences were annealed to their respective antisense complementary strands. Parallel control experiments were performed with the non–protein-binding B-PT sequence, 5'- AAAAGTCCCTTGCTGAAAGTC-3', or the non–protein-binding AP-1-PT sequence, 5'-CGCTTGACAGACTGGCCGGAA-3', and annealed to their respective complements. HUVEC monolayers were pretreated with 20 µmol/L ds-oligonucleotides for 3 hours before cells were treated with thiol agents.

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 1:5000 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% H₂O_2. The reaction was stopped by the addition of 75 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 the background values in cells stained only with the second-step antibody had been subtracted. All data points reflect triplicate values.

Preparation of Nuclear Extracts
HUVECs were plated on P-100 tissue culture dishes. Confluent HUVECs were exposed to diamide (0.2 mmol/L) for 15 minutes, washed, and then treated with BSO (1 mmol/L) for 4 hours in the absence or presence of ds-oligonucleotides that contained NF-B cognate DNA sequences. Parallel control experiments were performed in the absence of thiol agents. Nuclear extracts were prepared from control and treated cells by a modification of the method of Dignam et al.³⁴ 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 dithiothreitol, DTT). After recentrifugation, the cells were resuspended in 1 mL of hypotonic buffer that contained 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 supernatant (nuclear extract) that resulted was stored at -70°C. Protein concentrations were determined according to the Bradford³⁵ method. To minimize proteolysis, all buffers contained 0.2 mmol/L PMSF.

Electrophoretic Mobility Gel Shift Assays
³²P-radiolabeled ds-DNA probe for NF-B was generated from [-P]ATP and ds-oligonucleotides in a kinase reaction. The B ds-oligonucleotides used for the electrophoretic mobility shift assays 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 the manufacturer's specifications (Promega) at 37°C for 30 minutes in a buffer that contained 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), 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:bis ratio of 40:1) gels at 35 mA for 3 hours at room temperature in 0.5x Tris–boric acid EDTA buffer.³⁶ The gels were dried and autoradiographed.

Statistical Analysis
All values are expressed as mean±SEM. Data were analyzed with the use of a 1-way ANOVA with Bonferroni corrections for multiple comparisons or Fisher's protected least significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Figure 1 shows the effect of A/R on intracellular levels of GSH and GSSG/GSH ratio in HUVEC monolayers. Exposure of endothelial cells to 60 minutes of anoxia caused a significant decrease in cell GSH (Figure 1A) with concomitant increases in GSSG, which caused an increase in the GSSG/GSH ratio (Figure 1B). During reoxygenation, we found a further decrease in cell GSH that remained low during the 4-hour experimental period. Correspondingly, the GSSG/GSH ratio was significantly elevated for 4 hours. Figure 2 shows the kinetics of changes in the intracellular levels of GSH and GSSG/GSH ratio in HUVEC monolayers exposed to diamide plus BSO, diamide alone, or DEM plus BSO. Diamide at 0.2 mmol/L rapidly oxidizes cellular GSH to GSSG, which increases the GSSG/GSH ratio. With the addition of BSO, a low steady-state level of GSH and a high GSSG/GSH ratio were maintained for the duration of the experiment (6 hours). In the absence of BSO, the removal of diamide resulted in GSH resynthesis within 1 hour and the GSH thiol-disulfide status returned to control baseline values in 2 to 4 hours. Treatment of cells with DEM alone (0.5 mmol/L) significantly decreased cellular GSH at 15 minutes (2.4±0.4 nmol/mg protein versus 4.0±0.4 nmol/mg protein in control cells), but GSH recovered to untreated baseline values within 4 hours (4.1±0.1 nmol/mg protein). DEM treatment did not alter GSSG or GSSG/GSH ratio. To maintain a sustained low GSH during the time course of 6 hours, we coincubated cells with DEM and BSO (Figure 2). In summary, diamide plus BSO treatment induces a sustained high GSSG-to-GSH ratio for 15 to 360 minutes, diamide alone induces a transient increase in GSSG-to-GSH ratio at 15 to 30 minutes, but the thiol-disulfide status returned to control baseline values thereafter (90 to 360 minutes), whereas DEM plus BSO sustained low GSH levels throughout (15 to 360 minutes) without altering the GSSG/GSH status. These selected manipulations were used to delineate the inflammatory responses elicited by changes in GSSG/GSH ratio from those responses caused by changes in GSH.

View larger version (14K): [in this window] [in a new window]   Figure 1. Time course change of GSH and GSSG-to-GSH ratio in HUVECs exposed to 60 minutes of anoxia followed by 4 hours of reoxygenation. Each value indicates mean±SEM of 4 experiments. *P<0.05 and **P<0.01 vs control, preanoxic cells (ie, -60 minutes).

View larger version (28K): [in this window] [in a new window]   Figure 2. Time course of change in GSH (top) and GSSG-to-GSH ratio (bottom) in HUVECs treated with individual or combinations of chemical thiol agents. Each value indicates mean±SEM of 4 experiments. *P<0.05 and **P<0.01 vs 0 minutes.

Figure 3 shows the time course of neutrophil–endothelial cell adhesion elicited by treatment of HUVECs with the different thiol agents described in Figure 2. Incubation of HUVEC monolayers with diamide and BSO (Figure 3A) resulted in enhanced adhesivity of neutrophils, with peak adhesion responses at 15 minutes (phase 1) and 240 minutes (phase 2), which indicated that sustained alteration in cellular GSSG-to-GSH ratio elicited early- and late-phase inflammatory responses. Incubation of monolayers with diamide without BSO (Figure 3B) resulted in enhanced adhesivity of neutrophils to HUVECs with 1 peak adhesion response at 15 minutes (phase 1) but not at 240 minutes (phase 2), which showed that the phase 2 adhesion response is mediated by altered GSSG/GSH. Treatment of cells with DEM plus BSO also elicited a phase 1, but not a phase 2, neutrophil adhesion response (Figure 3C), which indicated that enhanced neutrophil adhesivity in the early phase is mediated by an altered GSH level that is independent of changes in GSSG.

View larger version (15K): [in this window] [in a new window]   Figure 3. Time course of neutrophil adherence to HUVECs after redox alteration. A, HUVECs treated with diamide plus BSO. B, HUVECs treated with diamide alone. C, HUVECs treated with DEM plus BSO. 51Cr-labeled neutrophils were added to HUVEC monolayers after removal of media that contained thiol agents, and neutrophil adherence was determined 30 minutes later. Each value indicates mean±SEM of 5 experiments performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 vs untreated controls.

Adhesion of neutrophils to endothelial cell monolayers was verified by phase-contrast microscopy, which showed significant adhesion of neutrophils to endothelial cells at 15 minutes and 240 minutes with diamide plus BSO, and at 15 minutes, but not at 240 minutes, with diamide alone or with DEM plus BSO (data not shown). Moreover, exposure of HUVEC monolayers to the various chemical agents for the specified duration (see Methods) without or with added neutrophils did not disrupt the monolayers, and the redox-altered HUVEC monolayers were similar in appearance to the redox-unaltered cell monolayers throughout the experimental period (data not shown). Furthermore, we observed no transmigration of adherent neutrophils through the endothelial monolayers in any of the experimental conditions.

To further assess the role of thiols in neutrophil–endothelial cell interaction, we pretreated redox-altered HUVEC monolayers with NAC. Figure 4 summarizes the effect of NAC on diamide plus BSO–induced neutrophil adherence at phase 1 and phase 2. The increase in neutrophil adherence to HUVECs was significantly reduced in both phases, which suggested that diamide plus BSO–induced neutrophil adherence was caused by perturbations of the intracellular GSH/GSSG status. To define the relationship between the early- and late-phase neutrophil adherence responses with endothelial cell GSH levels and the GSSG/GSH ratio, neutrophil adhesion was plotted as a function of either GSH or the GSSG/GSH ratio. The results in Figure 5 illustrate the respective relationships in the early and late adhesion responses. The data show that phase 1 neutrophil adhesion was highly and inversely correlated with GSH levels (r=0.87, P<0.001, Figure 5A), but not with the GSSG/GSH ratio (r=0.47, Figure 5B). In contrast, the phase 2 adhesion response was poorly correlated with GSH levels (r=0.51, Figure 5C), but was positively correlated with GSSG/GSH ratio (r=0.86, P<0.001, Figure 5D). These results are consistent with a predominant role for GSH in mediating the phase 1 adhesion response and for GSSG in mediating phase 2 adhesion.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of NAC on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Responses are shown for neutrophil adhesion at 15 minutes (phase 1) and 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 5 experiments performed in duplicate. *P<0.001 vs untreated controls; #P<0.01 vs diamide plus BSO treatment.

View larger version (25K): [in this window] [in a new window]   Figure 5. A and B, Relationship between phase 1 (15 minutes) neutrophil adherence and GSH level or GSSG/GSH ratio, respectively. C and D, Relationship between phase 2 (240 minutes) neutrophil adherence and GSH level or GSSG/GSH ratio, respectively.

Figure 6 summarizes the effects of MAbs directed against different ECAMs on neutrophil adherence to endothelial monolayers treated with thiol agents at phase 1 and phase 2. The increase in neutrophil adherence to HUVECs was significantly reduced in both phases by anti–P-selectin (PB1.3) and anti–ICAM-1 (8.4A6). The anti–E-selectin–specific antibody (CL3) significantly inhibited neutrophil adhesion to HUVECs in phase 2, but had no effect in phase 1, which suggested the participation of E-selectin in the late-phase inflammatory response.

View larger version (25K): [in this window] [in a new window]   Figure 6. Effect of MAbs to P-selectin (PB1.3, 20 µg/mL), E-selectin (CL3, 20 µg/mL), and ICAM-1 (8.4A6, 20 µg/mL) on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Responses are shown for neutrophil adhesion at 15 minutes (phase 1) and 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.001 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars).

To define the molecular determinants in endothelial cells in the early- and late-phase adhesion responses, we quantified surface expression of ECAMs. Figure 7 summarizes the results on surface expression of different ECAMs after 15 minutes and 240 minutes of treatment with diamide and BSO. The data show that P-selectin expression was significantly increased at 15 minutes (phase 1) and at 240 minutes (phase 2, Figure 7A). The expression of E-selectin, however, was only significantly elevated at phase 2 (Figure 7B), which is consistent with a role for this adhesion molecule in the late-phase response. Unlike P-selectin or E-selectin, the constitutive surface expression of ICAM-1 was high in phase 1 with an additional significant increase observed in phase 2 (Figure 7C).

View larger version (12K): [in this window] [in a new window]   Figure 7. Surface expression of ECAMs [P-selectin (A), E-selectin (B), and ICAM-1 (C)] in HUVECs incubated with diamide plus BSO (solid bars). Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.01 vs untreated controls (open bars).

The finding of an enhanced neutrophil adhesion response 4 hours after induction of the GSSG/GSH imbalance, coupled to the evidence implicating a role for E-selectin (which is not constitutively expressed on HUVECs), suggested that an elevated GSSG/GSH ratio elicits a transcription-dependent upregulation of ECAMs. To assess this possibility, we treated HUVEC monolayers with inhibitors of macromolecule synthesis, CHX (1 µg/mL) or ActD (2 µg/mL). The results summarized in Figure 8 demonstrate that inhibition of protein synthesis significantly attenuates neutrophil adherence in phase 2 but has no effect on the phase 1 adhesion response. These observations are consistent with a transcription-dependent late-phase response induced by redox imbalance.

View larger version (34K): [in this window] [in a new window]   Figure 8. Effect of inhibitors of translation (CHX) or transcription (ActD) on neutrophil adherence to HUVECs in phase 1 and phase 2 after diamide plus BSO incubation. Responses are shown for neutrophil adhesion at 15 minutes (phase 1) and 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.05 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars).

Previous studies from our laboratory and others have invoked a role for NF-B and AP-1 in A/R-, cytokine- and H₂O₂-mediated inflammatory reactions.^{1 15 18 19 20} To evaluate the contribution of these transcription factors to the enhancement of neutrophil adhesion induced by elevated GSSG/GSH, HUVEC monolayers were treated with a proteasome inhibitor (MG132, 5 µmol/L) or an inhibitor of AP-1 (3AB, 1 mmol/L). In other experiments, cells were treated with ds-phosphorothioate oligonucleotides (20 µmol/L each) as decoys for B and AP-1. The results summarized in Figure 9 show that blockade of NF-B or AP-1 activation by their respective chemical inhibitors (Figure 9A) or respective cognate DNA sequences (Figure 9B) significantly inhibited phase 2 neutrophil–endothelial cell interactions. To document that this enhancement of phase 2 neutrophil adhesion caused by increased GSSG/GSH ratio is associated with transcription-dependent upregulation of ECAM expression, we measured the surface expression of the different ECAMs in redox-altered HUVEC monolayers pretreated with decoy B or AP-1 ds-oligonucleotides (Figure 10). Upregulation of P-selectin and E-selectin surface expression induced by redox imbalance was abolished by cognate ds-oligonucleotide for B but not AP-1 (Figure 10A and 10B), which indicated that the transcription-dependent expression of these 2 adhesion molecules is specifically linked to NF-B, and not AP-1 activation. Interestingly, unlike P-selectin or E-selectin, the enhanced surface expression of ICAM-1 induced by elevated GSSG/GSH was significantly attenuated by blockade of both NF-B and AP-1 activation (Figure 10C), which indicated a role for both transcription factors in ICAM-1 expression.

View larger version (24K): [in this window] [in a new window]   Figure 9. Effect of inhibitors of NF-B or AP-1 on neutrophil adherence to HUVECs in phase 2 after diamide plus BSO incubation. A, Effect of MG132 (for NF-B) or 3AB (for AP-1). B, Effect of B or AP-1 ds-oligonucleotide decoys. Responses are shown for neutrophil adhesion at 240 minutes (phase 2) after redox change. Each value indicates mean±SEM of 3 or 4 experiments performed in triplicate. *P<0.05 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars).

View larger version (22K): [in this window] [in a new window]   Figure 10. Effect of B or AP-1 ds-oligonucleotides on diamide plus BSO–induced surface expression of ECAMs [P-selectin (A), E-selectin (B), and ICAM-1 (C)]. Non-B and non–AP-1 represent the respective nonbinding oligonucleotides. Each value indicates mean±SEM of 4 experiments performed in triplicate. *P<0.05 vs untreated controls (open bars); #P<0.05 vs diamide plus BSO treatment (solid bars).

To verify that the transcription-dependent phase 2 adhesion response and ECAM surface expression is associated with NF-B activation, we performed electrophoretic mobility shift assays (EMSAs) on nuclear extracts prepared from control cells and cells treated with thiol agents for 240 minutes. The results in Figure 11 show a specific B nucleoprotein adduct on gel shift (designated by filled arrow) that was eliminated with 50-fold excess of unlabeled oligonucleotide (lane 3). Treatment of cells with diamide and BSO for 240 minutes increased the amount of the NF-B–specific adduct (lane 2) compared with control (lane 1). Moreover, pretreatment of cells with the B decoy completely abolished binding of the adduct to the radiolabeled oligonucleotide (lane 4), which suggested that inhibition of NF-B activation by the decoy is specific. Nonbinding decoy had no effect on NF-B binding (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 11. EMSA on nuclear extracts from HUVECs incubated with diamide plus BSO in the presence or absence of B ds-oligonucleotide. The NF-B-specific adducts are indicated by bold arrow.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well appreciated that inflammatory reactions elicited by a variety of stimuli, such as H₂O₂, TNF, and A/R, are associated with induction of cellular oxidative stress via stimulation of ROS production.^{1 2 3} The contribution of redox imbalance, which often results from enhanced ROS formation, to the molecular events that lead to the inflammatory response is poorly understood. This study provides the first direct evidence to support the hypothesis that disruption of normal cellular thiol/disulfide status enhances neutrophil adhesion to endothelial cells by inducing the expression of different adhesion molecules on the endothelial cell surface. To delineate inflammatory responses elicited as a result of changes in the GSSG/GSH ratio from those caused by changes in GSH per se, we used 3 distinct treatment strategies to induce specific changes in either GSH alone or in both GSH and GSSG (Figure 2). Treatment with DEM and diamide alone caused transient disruption of cell GSH or GSSG that occurred within 15 to 30 minutes; thereafter, the redox status returned to baseline values. Treatment with DEM plus BSO resulted in a rapid and sustained decrease in endothelial cell GSH, but with no change in cell GSSG. A sustained cellular redox imbalance (decreased GSH and increased GSSG) was achieved with combined treatment of endothelial cell monolayers with diamide plus BSO. With these 2 agents, the kinetics of redox imbalance resemble those induced by exposure of endothelial cells to anoxia followed by reoxygenation.

Loss of GSH/GSSG balance caused an early (15 minutes, phase 1) and a late (4 hours, phase 2) neutrophil adhesion to HUVEC monolayers, which are similar to the kinetics of A/R-induced neutrophil adhesion.¹ The finding that NAC counters the neutrophil adherence induced by diamide plus BSO in both phases is consistent with a role for cellular thiol status in modulating neutrophil adhesivity to endothelial cells. Although the magnitude of the neutrophil adhesion responses in the 2 phases was similar, the contributions of specific ECAMs and thiol/disulfide status differ between the 2 phases. Our results show that phase 1 neutrophil adhesion was inversely correlated with GSH levels but not with GSSG/GSH ratio, whereas phase 2 adhesion response was positively correlated with GSSG/GSH ratio but not with GSH. These findings indicate that redox regulation of neutrophil–endothelial cell adhesion differs in a fundamental manner between the early and late responses to an oxidant stress.

The attenuating actions of monoclonal antibodies directed against either P-selectin or ICAM-1 on phase 1 adhesion suggest that these 2 adhesion molecules make a major contribution to the early neutrophil adhesion response, consistent with a role for the constitutively expressed ICAM-1³⁷ and the rapidly mobilizable pool of preformed P-selectin.^{38 39 40} The mechanism by which GSH modulates surface expression of ICAM-1 and P-selectin in phase 1 adhesion response is unclear. One possibility may be that cell GSH, much like cell ATP, regulates the dynamics of the microtubule assembly and thereby effects movement of P-selectin along microtubular tracks from storage Weibel-Palade bodies to endothelial cell surface.

In comparison, phase 2 adhesion is responsive to GSSG/GSH status and is mediated by E-selectin, P-selectin, and ICAM-1. The evidence implicating E-selectin and the finding that enhanced inflammatory response occurs 4 hours after induction of redox imbalance suggest that sustained disruption of thiol/disulfide homeostasis elicits transcription-dependent upregulation of ECAM. Our data demonstrate that treatment of HUVECs with inhibitors of transcription and translation or reagents that interfere with the activation of either NF-B or AP-1 inhibited phase 2 neutrophil adhesion and the expression of ECAMs. EMSAs confirmed that sustained increase in GSSG causes specific upregulation and activation of NF-B. Our previous studies have demonstrated a similar late-phase A/R-induced neutrophil–endothelial cell adhesion that involves activation of NF-B and transcription-dependent upregulation of E-selectin.¹

The mechanism by which increased GSSG modulates NF-B– and AP-1–dependent transcriptional upregulation of the different ECAMs is unclear. One possibility may be that GSSG promotes nuclear translocation of NF-B. Moreover, increased GSSG could promote binding of NF-B or AP-1 to their respective endogenous B or AP-1 enhancer-promotor elements in DNA. Recent studies have demonstrated that NF-B DNA binding activity in HeLa cell nuclear extracts was increased by H₂O₂.⁴¹ Thus, it appears that a highly oxidized environment, such as occurs with increased oxyradical formation and loss of GSH homeostasis, would favor activation of NF-B and enhance gene transcriptional activity. Another possible explanation for GSSG-induced activation of NF-B may relate to the action of diamide on free SH groups on proteins. Recent studies suggest that diamide may act as a protein tyrosine phosphatase inhibitor.⁴² In this regard, diamide can inhibit tyrosine phosphatase–catalyzed dephosphorylation via formation of protein disulfide cross-link in the phosphate transfer domain of the enzyme. Inhibition of phosphatase activity would result in hyperphosphorylation of I–B and subsequent activation of NF-B. These considerations underscore the potential importance of redox modulation of transcriptional activity of inflammatory genes, and delineation of the signaling mechanisms represents a current avenue of investigation in our laboratory. Regardless of mechanism, our present study provides strong evidence that redox perturbation leads to NF-B– and AP-1–dependent upregulation of adhesion molecules and enhanced neutrophil adherence to endothelial cells. A notable finding is that the enhanced expression of P-selectin and E-selectin elicited by redox imbalance appears to be mediated specifically by NF-B, in contrast to the involvement of NF-B as well as AP-1 in the transcriptional upregulation of ICAM-1.

The phase 2 transcription-dependent expression of E-selectin induced by redox imbalance is consistent with our previous results in the A/R model.¹ Interestingly, our present data show that enhanced P-selectin expression induced by GSH oxidation is predominantly transcription-dependent, which is in contrast to our previous observation that phase 2 surface expression of P-selectin elicited by A/R largely involves transcription-independent events. These results suggest that A/R-induced loss of redox balance mediates transcriptional upregulation of E-selectin, but redox regulation of P-selectin gene expression appears to have a lesser role during A/R. Notwithstanding, our finding that changes in GSSG/GSH levels can increase P-selectin gene transcription suggests that regulators of the transcriptional activity of this adhesion protein is under redox control. Although of less importance during A/R, this redox mechanism for P-selectin transcription may play a more prominent role in adhesion responses induced by other inflammatory stimuli such as cytokines, LPS, or hypercholesterolemia.

The contribution of endothelial cell redox imbalance to leukocyte activation is under investigation. Preliminary evidence reveals that leukocyte adhesion to redox-altered HUVEC monolayers in both phases was attenuated by the CD-18–specific MAb, which suggested upregulation of this leukocyte surface receptor. Our recent studies show that A/R-induced neutrophil adhesion to endothelial cells was similarly attenuated by anti–CD-18 MAb.^{1 21} Given the similarities in adhesion responses between redox-altered cells and cells subjected to A/R, we hypothesize that disruption of endothelial cell GSH/GSSG status, as was found to occur in A/R (Figure 1), would enhance production of platelet aggregating factor or platelet aggregating factor–like substances, which then mediate neutrophil activation.

In summary, our study shows that loss of thiol/disulfide balance can elicit neutrophil hyperadhesivity to endothelial cells. We propose that induction of redox change may be an early event that initiates a cascade of molecular signaling in neutrophil adhesion response during A/R. It should be noted that this proposal is based on results with the use of pharmacological agents to manipulate cellular thiol/disulfide status. Although these thiol agents have previously been well characterized with regard to GSH metabolism, we cannot rule out other potential nonspecific effects. However, we have used 3 separate thiol agents with different actions on GSH or GSSG, and the results are consistent with those observed with our previous A/R model¹ in terms of GSH and GSSG changes, kinetics of neutrophil–endothelial cell interactions, transcription-dependent expression of ECAMs, and activation of NF-B and AP-1. The correspondence of this redox model with the A/R model suggests that chemical induction of thiol/disulfide imbalance provides a reasonable in vitro model system to delineate redox control of cellular and molecular signaling mechanisms of A/R-induced leukocyte-endothelial cell interactions.

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (DK-44510 and DK-43785). Dr Aw is a recipient of an Established Investigatorship Award from the American Heart Association

Received July 6, 1998; accepted December 16, 1998.

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