Actin S-Nitrosylation Inhibits Neutrophil β2 Integrin Function*

The focus of this work was to elucidate the mechanism for inhibition of neutrophil β2 integrin adhesion molecules by hyperoxia. Results demonstrate that exposure to high oxygen partial pressures increases synthesis of reactive species derived from type 2 nitric-oxide synthase and myeloperoxidase, leading to excessive S-nitrosylation of β-actin and possibly profilin. Hyperoxia causes S-nitrosylation of the four cysteine moieties closest to the carboxyl-terminal end of actin, which results in formation of short actin filaments. This alters actin polymerization, network formation, and intracellular distribution, as well as inhibits β2 integrin clustering. If neutrophils are exposed to ultraviolet light to reverse S-nitrosylation, or are incubated with N-formyl-methionyl-leucine-phenylalanine to trigger “inside-out” activation, the effects of hyperoxia are reversed. We conclude that cytoskeletal changes triggered by hyperoxia inhibit β2 integrin-dependent neutrophil adhesion.

The goal of this work was to elucidate the mechanism for inhibition of neutrophil ␤ 2 integrin adhesion molecules by hyperoxia. Neutrophil-mediated host defense, as well as tissue injury in situations such as ischemia-reperfusion, involves neutrophil activation and recruitment to a vascular bed. These functions require integrin-mediated cell-cell and cell-extracellular matrix adhesion. Neutrophils express ␤ 1 , ␤ 2 , and ␤ 3 integrins, but the ␤ 2 family (known as CD18 integrins) is predominantly expressed, and ␤ 2 integrins play a central role in regulating neutrophil activation and endothelial adhesion (1).
When animals or humans are exposed to hyperbaric oxygen (HBO 2 ) 3 at 2.8 -3.0 atmospheres absolute (ATA), the ability of circulating neutrophils to adhere to target tissues is temporarily inhibited (2)(3)(4)(5)(6). In animal models, HBO 2 -mediated inhibition of neutrophil ␤ 2 integrin adhesion has been shown to ameliorate reperfusion injuries of brain, skeletal muscle, and intestine as well as smoke-induced lung injury, decompression sickness, and encephalopathy because of carbon monoxide poisoning (2, 6 -13). Impaired neutrophil adherence by HBO 2 may be the basis for benefits shown in human clinical trials, including reductions in coronary artery re-stenosis after balloon angioplasty/stenting (14,15), decreased muscle loss after thrombolytic treatment for myocardial infarction (16,17), and reduced incidence of encephalopathy seen after cardiopulmonary bypass and carbon monoxide poisoning (18,19). Previous studies have shown that HBO 2 does not reduce neutrophil viability or cause immunocompromise, and functions such as degranulation and oxidative burst in response to chemoattractants appear to be intact (2,3).
Integrins are normally poorly adhesive, and their activation involves modifications in the conformation of the extracellular components to increase the affinity for binding and a coordinated grouping of integrins in the plane of the membrane to increase their avidity. Conformational (or affinity) changes and clustering (or avidity) represent two distinct events that appear to operate in a complementary manner to control cell adhesion (20,21). The actin cytoskeleton is involved with both of these events. Integrins can be activated by intracellular mechanisms (inside-out path) and by extracellular contacts (outside-in path), thus making the integrin-actin complex a communication conduit. Actin binding via an assortment of linking proteins provides a mechanical platform that brings integrins and activable enzymes together (22,23). There are over 50 proteins involved with connecting integrins and the actin cytoskeleton (24).
The goal of this investigation was to elucidate the mechanism for the inhibitory effect of HBO 2 on neutrophil ␤ 2 integrin function. We hypothesized that the HBO 2 mechanism was related to production of reactive species. Production of superoxide radical (O 2 ⅐ ) and hydrogen peroxide (H 2 O 2 ) is increased by hyperoxia, and HBO 2 can also increase nitric oxide ( ⅐ NO) production by stimulating isoforms of nitric-oxide synthase (NOS) (25)(26)(27). In this study we show that neutrophil NOS and myeloperoxidase (MPO) are required for ␤ 2 integrin inhibition and that cytoskeletal protein S-nitrosylation is a result. Filamentous actin (F-actin) formation, reduced CD18 clustering, and a subtle perturbation in the association between profilin and cytoskeletal actin occur in response to hyperoxia. Incubation with the chemotactic peptide, N-formyl-methionyl-leucylphenylalanine (fMLP), which modulates actin protein binding by changing cellular phosphoinositides via the so-called insideout path, can override the HBO 2 effect (28,29). Ultraviolet light (UV) photo-reversal of protein S-nitrosylation also restores ␤ 2 integrin function. Thus, HBO 2 inhibits neutrophil ␤ 2 integrin activation by generating reactive nitrogen species that perturb the cytoskeleton.
Animals-Wild type mice (Mus musculus) and MPO knockout mice were purchased (The Jackson Laboratories, Bar Harbor, ME), fed a standard rodent diet and water ad libitum, and housed in the animal facility of the University of Pennsylvania. A colony of MPO knock-out mice was maintained from breeding pairs purchased from The Jackson Laboratories. Male Wistar rats were purchased from Harlan Laboratories (Indianapolis, IN). Mice and rats were exposed to O 2 at 1-3 ATA for 45 min following our published protocol (30). After anesthesia (intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg)), skin was prepared by swabbing with Betadine, and the blood was collected into heparinized syringes following aortic puncture.
Isolation of Neutrophils-Neutrophils were isolated from heparinized blood using a protocol modified from Fekete et al. (31). Blood was made to a volume of 2.5 ml with cell buffer (PBS containing 1 mM CaCl 2 , 1.5 mM MgCl 2 , and 5.5 mM glucose) that was placed over 5 ml of Robbins PMN Prep plus 0.4 ml of 1.5% NaCl in a 15-ml silicone-coated glass tube and centrifuged at 400 ϫ g for 30 min. The platelet-rich plasma was discarded; the mononuclear cell layer was obtained on occasion, and the underlying neutrophil layer was always collected. The neutrophils and at times the monocytes were placed in silicone-coated glass tubes and combined with an equal volume of 0.45% NaCl for 5 min. The cells were then washed by adding cell buffer at a 3:1 ratio and centrifuging at 200 ϫ g for 10 min. The supernatant was discarded, and cell pellets were resuspended in NH 4 Cl/ Tris Lysis Buffer for 10 min. The solutions were centrifuged at 450 ϫ g for 10 min, and the cells were resuspended in cell buffer at a concentration of 5 ϫ 10 6 /ml. Final isolations were greater than 93% pure with greater than 98% live cells as shown by trypan blue exclusion.
Fibrinogen-coated Plate Adherence-Fibrinogen-coated plates were prepared by washing 15-mm Petri plates with H 2 SO 4 and rinsing three times with distilled water. Fibrinogen (500 l of solution containing 30 g/ml PBS) was added to each plate. After a 3-h incubation at 37°C, the plates were rinsed twice with PBS and were ready for use. Isolated neutrophils exposed to air or hyperoxia ex vivo, or simply taken from animals exposed to air or HBO 2 , were incubated for 30 min at 37°C with calcein AM (5 g/ml), washed twice with cell buffer, and then resuspended to a count of 5 ϫ 10 5 /100 l. Aliquots of 100 l were incubated on plates either alone, with anti-CD18 antibody (4 g/ml) for 10 min at room temperature, with 100 nM fMLP for 1 min, or with anti-CD18 antibody for 10 min followed by 100 nM fMLP for 1 min. After incubation, 100 l of buffer was removed from the plates, and the plates were washed twice with 4 ml of PBS to remove nonadherent cells. Fluorescence on the plates was measured (480 nm excitation/520 nm emission) in a plate reader. Adhesion was always assessed in triplicate and calculated as fluorescence in washed wells versus fluorescence in wells where cells were added but not washed to remove nonadherent cells. In each experiment specificity for ␤ 2 integrin adherence was checked by inclusion of a cell set incubated with anti-CD18 antibody, which reduced adherence to 0 -2%. In a sub-set of experiments, prior to placement on plates, neutrophils were exposed for 5 min to UV light from a 200-watt mercury vapor lamp kept at sufficient distance so that temperature was not altered.
Flow Cytometry-Flow cytometry was performed with a 4-color, dual laser analog FACSCalibur (BD Biosciences) using CellQuest acquisition software, or 18-color LSRII (BD Biosciences) using FACsDiva digital acquisition electronics and software (BD Biosciences) by standard protocol. Fluorophore (fluorescein isothiocyanate, RPE, or APC)-conjugated goat anti-mouse IgG and unstained cells served as negative controls. Compensation for 4-color staining samples was concentrationdependent and was determined empirically. Compensation for multicolor assays was performed automatically by FACSDiva software on the LSRII. Identification and enumeration of ICAM beads, ␤-integrins, and neutrophils were performed using CellQuest TM acquisition/analysis software (BD Biosciences), including three-dimensional histograms for Quantum Plex TM SP microbeads quantification and the FlowJo Program (Tree-Star Inc., Ashland, OR).
Surface Expression of ␤ 2 Integrins-After mice were exposed to HBO 2 , neutrophils were obtained and incubated with 4 l of anti-CD18 RPE-conjugated antibody and incubated at room temperature for 5 min. The samples were divided into 2 aliquots, and 100 nM fMLP was added to 1 aliquot and an equal volume of buffer was added to the other. After incubation for 1 min at room temperature, 1.5 ml of cold HBSS buffer was added to all of the tubes, and samples were centrifuged at 800 ϫ g for 5 min, washed twice with 1 ml of cold HBSS, then resuspended in HBSS, and assayed in the flow cytometer. Forward scatter and side scatter was used to isolate neutrophil populations, and the mean linear fluorescence was examined for the different samples. In some trials, assurance pertaining to accurate neu-trophil gating location was verified by co-labeling cells with anti-CD66 or anti-Ly-6G (32,33). These extra manipulations did not modify ␤ 2 integrin expression.
Oxidative Burst-After isolation, neutrophils were incubated with 5 M DCF-diacetate at 37°C for 15 min. Samples were divided into 2 aliquots, and 100 nM fMLP was added to one and HBSS buffer was added to the other followed by incubation for 1 min at room temperature. Then 1.5 ml of cold HBSS buffer was added to all of the tubes; samples were centrifuged at 800 ϫ g for 5 min, washed twice with 1 ml of cold HBSS, and then resuspended in HBSS for flow cytometry analysis of DCF fluorescence of neutrophils that were identified by forward and side laser light scatter and anti-Ly-6G staining.
Fluorescent Bead Adhesion Assay-ICAM-coated beads were prepared by incubating anti-mouse IgG-linked Quantum Plex TM SP beads (Bangs Laboratories, Fishers, IN; 50 l, ϳ1 ϫ 10 5 red-stained 4.4 m beads that fluoresce in FL3 channel) with 50 l of anti-human Fc receptor mouse IgG and incubated for 30 min at room temperature. Suspensions were centrifuged at 1000 ϫ g for 10 min, washed twice, and resuspended in 100 l of PBS. Then 25 g of mouse ICAM-1/human Fc chimera was added and incubated for 1 h at room temperature, followed by 20 l of mouse serum to block residual Fc receptors, and incubated for an additional 30 min. The bead complex was washed three times with PBS, resuspended in 200 l of PBS, and stored at 4°C until use. Isolated neutrophils from control or HBO 2exposed mice were counted and incubated with beads at a ratio of ϳ20 beads/cell in the absence or presence of 100 nM fMLP. Adhesion was determined by flow cytometry.
Microelectrode Assay-Production of ⅐ NO by cells exposed to various partial pressures of O 2 was carried out using published procedures (26,34). In brief, neutrophils were suspended in cell buffer at a concentration of 2.5 ϫ 10 6 /ml and placed in a hyperbaric chamber with pass-through connections where a polarographic ⅐ NO meter (ISO-NOP200, World Precision Instruments, Sarasota, FL.) had been mounted and calibrated using authentic ⅐ NO. Cells were continuously stirred while exposed to air, and the current was recorded. While maintaining the cells and apparatus under the same operating conditions, the chamber was flushed with pure O 2 and the signal re-recorded. The chamber was then slowly pressurized to 2.0 and then 3.0 ATA. Each change in pressure was carried out slowly so that ambient temperature changed less than 2°C, and the current was recorded only after buffer temperature had returned to room temperature (21°C). Using the same cell suspension for each partial pressure of O 2 , the difference in responses was compared. The study was then repeated, but cells were suspended in buffer containing 1 mM N G -nitro-L-arginine methyl ester (L-NAME). The difference in signals Ϯ1 mM L-NAME was taken as ⅐ NO production by the cells.
Cell Extract Preparation and Biotin-switch Assay-Neutrophils were isolated from control and HBO 2 -exposed mice and resuspended in 2 ml of HEN buffer (250 mM Hepes, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine), sonicated on ice for 30 s, and then passed through a 28-gauge needle five times. Lysates were centrifuged at 2000 ϫ g for 10 min, and the supernatant was recovered and made to 0.4% CHAPS using a 10% stock solution. The biotin-switch assay was carried out essentially as described by Jaffrey et al. (35). After adding 4 volumes of blocking buffer (HEN buffer ϩ 2.5% SDS, 20 mM methyl methanethiosulfonate), samples were incubated at 50°C for 20 min with frequent vortexing. Ten volumes of Ϫ20°C acetone was added and incubated at Ϫ20°C for 20 min, followed by centrifugation at 2000 ϫ g for 10 min and resuspension in 0.1 ml of HENS buffer ϩ 1% SDS/mg protein. Biotinylation was accomplished by adding a 1:3 volume of labeling solution (4 mM biotin-HPDP dissolved in dimethylformamide) and a 1:50 volume of 50 mM ascorbate followed by incubation for 1 h at 25°C. In selected trials, biotinswitch analysis was performed on cell lysates treated with HPDP-biotin or with ascorbate (but not both) or with 1 mM HgCl 2 for 10 min at room temperature (which cleaves S-NO bonds).
For initial identification of nitrosylated proteins, 2 volumes of Ϫ20°C acetone were added to lysate samples and incubated at Ϫ20°C for 20 min, and samples were centrifuged at 2000 ϫ g for 10 min. The supernatants were discarded and pellets resuspended in SDS buffer for Western blots that were probed with anti-biotin following published methods (36). Band densities were quantified using an Odyssey Infrared Imager (Li-Cor, Inc., Lincoln, NE). When samples were used for protein digestion followed by affinity peptide capture, cell pellets were resuspended in 5 ml of incubation buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.4% Triton X-100), 1:20 endoproteinase GluC (V8), and incubated at room temperature for 16 h in the dark. It should be noted that early trials performed using 1:100 diethylpyrocarbonate-treated trypsin did not recover HPDP-modified peptides. After the desired protease digestion, samples were made to 0.5 mM PMSF and passed through Ultra-free-MC 10-kDa cutoff filters there were previously rinsed with methanol and washed with water. The filtrate was recovered and incubated with 50 l of dry, washed streptavidin-Sepharose beads for 30 min. After centrifugation at 5,000 ϫ g for 5 min, beads were washed five times in 10 volumes of 1 M ammonium carbonate, followed by five washes with 10 volumes of deionized water. Peptides were eluted by incubating beads with buffer at pH 3.8 for 30 min. After centrifuging beads at 5,000 ϫ g for 4 min, the supernatant was removed, and peptide fragments were eluted with 70% formic acid and evaporated to ϳ5 l in vacuo, followed by resuspension in 20 l of PBS.
Protein and Peptide Analysis-Mass spectrometry (MS) and protein sequencing were done by the Proteomics Core Facility of the Genomics Institute and the Abramson Cancer Center, University of Pennsylvania. Protein bands identified as containing biotin on Western blots were cut from nitrocellulose paper, trypsin-digested, and analyzed with a nano-liquid chromatography/nanospray/LTQ mass spectrometer following our published methods (36). The raw data files were searched using Mascot against the NCBI data base with a cutoff protein score of 70. For analyzing the peptides obtained following affinity purification with streptavidin-Sepharose beads, peptides were dissolved in 10 l of 0.1% formic acid ϩ 1% acetonitrile and 3 l injected and separated with a C18 column at a flow rate of 200 nl/min. The separated peptides were eluted and sprayed on line into the LTQ (Thermo Electron) at 22 kV. The raw data from MS/MS spectra were acquired with Xcalibur and searched with Sequest. Only peptide assignments with a differential 428-atomic mass unit addition to cysteine because of HPDPbiotin in the y-or b-ion series were considered. The crosscorrelation (xC) score cutoff was 1.5 for single charged, 2.0 for double charged, and 2.5 for triple charged peptides.
Filamentous Actin and CD18 Imaging-Neutrophils isolated from control and HBO 2 -exposed mice were placed on glass slides that had been coated with fibrinogen. Poly-L-lysinecoated slides were incubated with a solution of fibrinogen (200 g/ml) for 3 h at 37°C and then cooled to room temperature prior to use. An 80-l cell suspension was incubated on slides for 10 min and then fixed by addition of 80 l of cold 4% paraformaldehyde in PBS, followed by incubation for 30 min at 4°C. Autofluorescence was quenched by incubation in ice-cold PBS containing 0.5% bovine serum albumin and 50 mM NH 4 Cl for 15 min and then rinsed with PBS. Cells were then permeabilized by adding 2 mM palmitoyl L-␣-phosphatidylcholine along with 1 unit/ml of Alexa Fluor 488 phallacidin (Molecular Probes) and 1:1000 mouse anti-CD18-RPE. Following incubation in the dark for 30 min at 37°C, slides were washed five times with cold HBSS, and coverslipped. Examinations were carried out with a Bio-Rad Radiance 2000 attached to a Nikon TE 300 inverted stage confocal microscope that was operated with a red diode laser at 638 nm, and krypton lasers at 488 and 543 nm. Quantification of the fluorescence signals was performed using MetaMorph Image Analysis software (MDS, Inc., Toronto, Canada).
Immunoprecipitation of Protein Complexes after in Situ Cross-linking-Cell suspensions from control or HBO 2 -exposed mice were placed on fibrinogen-coated plates, and after 10-min incubations, adherent cells were subjected to protein cross-linking essentially as described by Huttelmaier et al. (37) using dithiobis(succinimidyl propionate) (DTSP) (Pierce). DTSP cross-links proteins over a span of ϳ12 Å (37). After 300 l of PBS containing 0.5 mM DTSP was added to the adherent cells, they were incubated for 30 min at room temperature. Plates were washed twice with PBS and then with PBS ϩ 0.2 M glycine to quench cross-linking. After two additional washes, 0.5 ml of RIPA buffer was added (50 mM Tris, pH 7.2, 150 mM NaCl, 20 mM glycine, 2.5 mM sodium azide, 1 mM EGTA, 1% Triton X-100, 0.25% deoxycholate, 1 g/ml leupeptin, 0.5 g/ml aprotinin, and 1 mM PMSF) and incubated on ice for 30 min. The cells were scraped off the plates with a rubber policeman and centrifuged at 15,000 ϫ g for 10 min, and the supernatant was collected. A pre-cleared supernatant volume containing 250 g of protein was incubated with 5 g of anti-actin antibodies on a shaker overnight at 4°C, and then 30 l of 20% (w/v) protein G-Sepharose (pre-blocked with 2% bovine serum albumin) was added and incubated for 1.5 h at 4°C. Samples were washed twice in RIPA buffer, pelleted, suspended in 20 l of heated SDS buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 20% ␤-mercaptoethanol), and incubated at 95°C for 15-20 min. Proteins were subjected to electrophoresis in 12% SDSpolyacrylamide gels, and Western blot analysis was performed as described previously (36).
Cytoskeletal Protein Analysis Based on Triton Solubility-Neutrophil suspensions from control or HBO 2 -exposed mice were placed on fibrinogen-coated plates, and after 10-min incubations, adherent cells were processed essentially as described by Yan and Berton (38). Cytoskeleton stabilization buffer (CSK, 300 l) (25 mM Hepes, pH 6.9, 0.2% Triton X-100, 1 M glycerol, 1 mM EGTA, 1 mM PMSF, 1 mM MgCl 2 ) was added to the plate, incubated for 10 min at room temperature, and removed for use as the Triton-soluble cell protein fraction. Where indicated, this solution was centrifuged at 366,000 ϫ g for 2 min to separate short actin filaments from G-actin following published methods (39). Residual Triton-insoluble proteins that remained on the plates were dissolved with SDS buffer heated to 95°C and incubated on the plates for 20 min. Both the Tritonsoluble and Triton-insoluble proteins were subjected to electrophoresis in 12% SDS-polyacrylamide gels and Western blotting.
Ex Vivo Actin Polymerization-The kinetics of actin polymerization was measured using pyrene-labeled ␣-actin from rabbit skeletal muscle following procedures provided by the supplier (Cytoskeleton, Inc., Denver, CO). The basis of this assay is that the fluorescence intensity of pyrene-labeled polymeric actin is much greater than for monomeric actin. Polymerization was induced in solutions (200 l) containing 10 M pyrene G-actin and 50 g of lysate protein prepared from neutrophils suspended in CSK buffer by adding 0.1 volume of buffer containing 500 mM KCl, 20 mM MgCl 2 , and 10 mM ATP. Studies were performed at room temperature, and the linear change in fluorescence was recorded using an excitation wavelength of 365 nm and emission wavelength of 407 nm.
Viscometry-Falling ball viscometry was assessed following published methods (40). Solutions were prepared as in the polymerization assay using 25 M purified rabbit G-actin. All solutions were kept at 4°C in the dark until use. Samples were loaded into 0.2-ml glass pipettes with internal diameters of ϳ1.5 mm, placed in a platform to hold them at a 60 o angle to the horizontal, and stored in the dark at room temperature. Preliminary trials demonstrated that the maximum viscosity was reached after 12 h, so all studies were performed at that time. The apparent viscosity of the sample was assessed by placing a 1-mm stainless steel ball (McMaster-Carr, Chicago, IL) into the pipette and measuring the rate of fall through the solution column. Viscosity was expressed as the reciprocal of the velocity of the falling ball (s/cm).
Statistical Analysis-Results were expressed as the means Ϯ S.E. for three or more independent experiments. To compare data, we used a one-way analysis of variance using SigmaStat (Jandel Scientific, San Jose, CA) and Newman-Keuls post-hoc test. The level of statistical significance was defined as p Ͻ 0.05.

RESULTS
Fibrinogen Plate Adhesion-Adherence because of ␤ 2 integrins was assessed using neutrophils obtained from the blood of mice exposed only to air (control), or after mice were exposed for 45 min to 100% O 2 (1 ATA) or to 2.8 ATA O 2 (HBO 2 ). As in previous studies, 1 ATA O 2 caused no significant alteration in ␤ 2 integrin function (adherence ϭ 19.4 Ϯ 3% (S.E., n ϭ 4)), but there was a significant reduction in adherence of neutrophils from mice exposed to HBO 2 (Fig. 1). We found that although adhesion was inhibited for neutrophils, no significant inhibition was observed for monocytes obtained from the same animals.
As we hypothesized that free radical production was the basis for the HBO 2 -mediated effect, we examined adherence of neutrophils from mice that had been injected with the NOS inhibitor L-NAME. As shown, neutrophils from these animals exhibited normal adherence to fibrinogen. The predominant NOS isoform in neutrophils is the inflammatory or type 2 (iNOS) that can be inhibited specifically with 1400W (N-(3-(aminomethyl)benzyl) acetamine). If mice were injected with 2 mg/kg 1400W for 30 min before HBO 2 exposure, neutrophil adherence to fibrinogen-coated plates was 27.8 Ϯ 2.5% (S.E., n ϭ 6). This value was insignificantly different from the value with airbreathing control mice injected with 1400W following the same protocol, where neutrophil adherence was 30.8 Ϯ 3.6% (S.E., n ϭ 6). The inhibitory effect of HBO 2 could also be resolved if 0.1 M fMLP was added to the wells of fibrinogencoated plates. Thus, rather than observing 3.0 Ϯ 0.9% adherence (HBO 2 effect), adherence was 30.3 Ϯ 7.3% (n ϭ 4), which was insignificantly different from adherence observed for cells from air-exposed, control mice incubated with fMLP, 27.5 Ϯ 7.0% (n ϭ 4).
Given that the HBO 2 inhibitory effect appeared to be discrete for neutrophils, we next examined ␤ 2 integrin-dependent adherence of neutrophils from MPO knock-out mice. As shown (Fig. 1), no inhibition of adhesion function was observed.
Next, neutrophils were isolated from air-breathing control mice and cells exposed for 45 min to various partial pressures of O 2 . As shown in Fig. 2, ex vivo exposure to hyperoxia also inhibited adherence. If air-only exposed cells or HBO 2 -exposed cells were incubated with anti-CD18 antibodies to block ␤ 2 integrins, adherence was 1 Ϯ 2%, indicating that adhesion to fibrinogen-coated plates was entirely dependent on ␤ 2 integrins. These results are consistent with in vivo O 2 exposure studies because when animals breathe 100% O 2 at 1 ATA O 2 , circulating cells briefly are exposed to ϳ720 mm Hg, but for the majority of time they experience O 2 tensions closer to that in the capillary (ϳ40 mm Hg). When humans or animals are exposed to 1 ATA O 2 , tissue oxygenation changes marginally, and only with ventilation at 2-3 ATA O 2 does tissue O 2 tension rise to levels of ϳ300 -600 mm Hg (41). Therefore, ex vivo exposure to 1 ATA O 2 is close in magnitude to the oxygenation cells experience in vivo when animals and humans are exposed to HBO 2 .
Surface Expression of CD18-The surface expression of ␤ 2 integrins on neutrophils from control or HBO 2 -exposed mice was assessed by flow cytometry. As shown in Table 1, HBO 2 did not inhibit CD18 expression. In fact, cells from mice exposed to HBO 2 exhibited significantly more CD18 on the cell surface than control cells. Surface expression was significantly increased when cells were incubated with 0.1 M fMLP in both control and HBO 2 -exposed cells.
Oxidative Burst-Oxidative burst was evaluated in neutrophils from control or HBO 2 -exposed mice. As shown in Table 2, cells from HBO 2 -exposed mice exhibited significantly greater fluorescence than control cells, and fMLP significantly increased DCF fluorescence in both control and HBO 2 -exposed cells. Adherence of neutrophils exposed to various O 2 partial pressures for 45 min ex vivo. Neutrophils were isolated from blood, suspended in PBS containing 1 mM CaCl 2 , 1.5 mM MgCl 2 , and 5.5 mM glucose, and exposed for 45 min to air or O 2 at 1, 2, or 2.8 ATA prior to calcein AM incubation assessment of adherence as described under "Experimental Procedures." Data are means Ϯ S.E. *, p Ͻ 0.05.

TABLE 1 CD18 surface expression on neutrophils
Neutrophils were isolated from air-breathing control mice or from mice first exposed to 2.8 ATA O 2 for 45 min. After incubating cells with anti-CD18 antibodies, surface expression of CD18 was assessed by flow cytometry as described under "Experimental Procedures." Where indicated, cells were incubated with 100 nM fMLP prior to analysis. Data are means Ϯ S.E.

PBS PBS ؉ fMLP
Control ( Adherence of ICAM-coated Microspheres-The affinity of ␤ 2 integrins was assessed by incubating neutrophils with fluorescent microspheres coated with ICAM-1 (Fig. 3). Fluorescence was significantly greater in HBO 2 -exposed cells versus cells from control mice, and co-incubation with fMLP significantly increases microsphere adherence in both control and HBO 2exposed cells. Mean fluorescence, however, does not depict the entire effect of HBO 2 . When data are displayed to the threedimensional contour plots, it becomes clear that HBO 2 -exposed cells exhibit more intracellular complexity even before fMLP exposure.
⅐ NO Production during Hyperoxia-The ability of L-NAME and 1400W to reverse the HBO 2 -mediated effect on adhesion suggests that cells generate ⅐ NO in response to hyperoxia. Therefore, we wanted to evaluate this directly by measuring ⅐ NO production in neutrophils exposed to different partial pressures of O 2 . Initial trials with mouse cells met with indeterminant results because of the rather low yields of cells from the average mouse. We have previously reported that HBO 2 has the same inhibitory effect on neutrophils from rats and humans as we see in mice (2,3). Therefore, to obtain more cells for comparisons across different O 2 partial pressures, neutrophils were obtained from rats, and ⅐ NO production was assessed using cell suspensions exposed to air or pure O 2 at 1, 2, or 3 ATA. Results in Fig. 4 demonstrate that HBO 2 does indeed stimulate ⅐ NO synthesis by neutrophils.
Protein S-Nitrosylation-Finding that MPO and NOS activity was required for HBO 2 -mediated inhibition of ␤ 2 integrins provides important insight into potential mechanisms. MPOdependent metabolism of nitrite and peroxide can generate ⅐ NO-derived oxidants capable of nitration and S-nitrosylation reactions. We failed to find excess nitrotyrosine in neutrophils from HBO 2 -exposed mice using dot-plot assays of whole cell homogenates or by Western blot (not shown). Therefore, we sought evidence for S-nitrosylation of neutrophil proteins by the biotin-switch assay, which covalently adds a disulfidelinked biotin to the labile S-nitrosylation sites on proteins.
Western blots prepared from control and HBO 2 -exposed neutrophils were probed with anti-biotin antibodies, and three bands were visualized (Fig. 5). In separate trials, the three bands were cut from nitrocellulose paper, subjected to amino acid sequencing, and tentatively identified as actin, a partially degraded actin fraction, and profilin. If the biotin-switch analysis was performed on cell lysates treated with HPDP-biotin or with ascorbate (but not both), with 1 mM HgCl 2 , or if cells were exposed to UV light prior to cell lysis and biotin-switch, the bands were not visualized (not shown). As shown in Fig. 5, the density of biotin-containing bands was doubled in the HBO 2exposed neutrophils, but there were no differences in band density between control and HBO 2 -exposed monocytes, neutrophils obtained from mice pre-treated with L-NAME, or in neutrophils from MPO knock-out mice.
Results in Fig. 6 demonstrate that ␤-actin and profilin band densities were not different from control if neutrophils from HBO 2 -exposed mice were incubated with 0.1 M fMLP or if FIGURE 3. ICAM bead adherence for integrin avidity assessment. Neutrophils were isolated from air-breathing control mice or after exposure to 2.8 ATA O 2 for 45 min. Isolated cells were then incubated with fluorescent beads coated with mouse ICAM as described under "Experimental Procedures," and binding was assessed by flow cytometry. Where indicated 100 nM fMLP was added 1 min before mixing cells with ICAM beads. Data in the table below are mean channel fluorescence Ϯ S.E. for three trials using cells from different animals. *, p Ͻ 0.05.

TABLE 2 Oxidative burst (DCF) from neutrophils
Neutrophils were isolated from air-breathing control mice or from mice first exposed to 2.8 ATA O 2 for 45 min. After incubating cells with 5 M DCF-diacetate for 15 min, fluorescence was assessed as described under "Experimental Procedures." Where indicated, cells were incubated with 100 nM fMLP 1 min prior to analysis. Data are means Ϯ S.E.

PBS PBS ؉ fMLP
Control (  Because the pico-amperage output detected in complex suspensions may not be due entirely to ⅐ NO, results are expressed as the difference in the signal between identical cell suspensions incubated in the absence and presence of 1 mM L-NAME to more reliably attribute findings to NOS activity. A standard curve was generated using ⅐ NO in HBSS. Data are mean Ϯ S.E., n ϭ 5. *, p Ͻ 0.05. exposed to UV light prior to lysis and biotin-switch analysis. In keeping with these findings, we also examined whether UV light exposure would reverse the HBO 2 -mediated inhibition of ␤ 2 integrin neutrophil adhesion. Neutrophils from control mice exposed to UV light following the same protocol that reversed the biotin-switch findings exhibited 26.8 Ϯ 1.2% (n ϭ 4, S.E.) adherence to fibrinogen-coated plates, whereas neutrophils from HBO 2 -exposed mice exhibited 26.9 Ϯ 4.2% (n ϭ 4, no significant difference).
We wished to determine the fraction of ␤-actin and profilin that were S-nitrosylated in neutrophils because of HBO 2 . Cells were isolated from air-exposed, control, and HBO 2 -exposed mice, and lysates were prepared and subjected to the biotinswitch assay. Biotin-bound proteins were affinity-captured with streptavidin-Sepharose beads, and Western blots were prepared using supernatant (nonaffinity-captured) proteins and those precipitated by streptavidin beads. In cells from airexposed mice, streptavidin beads precipitated 27.7 Ϯ 4.3% (n ϭ 3, S.E.) of actin and 40.6 Ϯ 1.2% (n ϭ 3, S.E.) of cell profilin. In cells from HBO 2 -exposed mice, bead treatment precipitated 68.7 Ϯ 3.7% (n ϭ 3, p Ͻ 0.05) of cell actin and 58.2 Ϯ 2.4% (n ϭ 3, p Ͻ 0.05) of cell profilin.
Actin Nitrosylation in Control and HBO 2 -exposed Neutrophils-To survey for nitrosylated proteins in an alternative manner, neutrophil lysates were incubated with HPDPbiotin and ascorbate following the standard biotin-switch protocol and subjected to proteolysis and peptide fragments affinity-captured with streptavidin beads. The criteria for evaluation and acceptance of peptide MS/MS spectra are described under "Experimental Procedures." HPDP-biotin containing peptide fragments were only found for mouse ␤-actin (Fig. 7), and the spectra for these fragments are shown in the supplemental material. Nitrosylation of the two cysteine moieties closest to the carboxyl-terminal end of the ␤-actin protein occurred in control neutrophils. Actin from HBO 2 -exposed neutrophils exhibited nitrosylation at these same two cysteine sites, and also at the next two cysteine moieties in the protein chain, i.e. at the four cysteine sites closest to the carboxyl-terminal end of the protein.
F-actin and Integrin Clustering-The biochemical analysis of neutrophils implicates alterations of ␤-actin as the cause for HBO 2 -mediated inhibition of ␤ 2 integrin adherence. To gain further insight into the consequences of actin S-nitrosylation, cells were placed onto fibrinogen-coated slides and permeabilized to allow imaging of filamentous actin (F-actin). Fig. 8 demonstrates the profound effect HBO 2 has on F-actin formation assessed by phallacidin binding. In the same cells we also probed ␤ 2 integrin clustering with RPE-conjugated anti-CD18. Fig. 9 shows quantitative changes in F-actin and inhibition of HBO 2 -mediated F-actin formation if neutrophils were incubated with fMLP or exposed to UV light for 5 min before plating onto slides. Fluorescence related to ␤ 2 integrin clustering was also restored if neutrophils were incubated with fMLP or exposed to UV light.
Immunoprecipitation following Protein Cross-linking-To evaluate protein associations with ␤-actin, neutrophils adherent to fibrinogen plates were treated with the membrane-permeable protein cross-linker DTSP and lysed 30 min later. Following actin immunoprecipitation, blots were analyzed looking for differences in proteins band densities. We reasoned that by comparing band patterns between cells kept in the dark versus cells exposed to UV light for 5 min prior to DTSP treatment, the differences in protein associations with ␤-actin because of HBO 2 may be identified. The stained gel image in Fig. 10 is representative of the results from six replicate experiments. Although there was a complex band pattern, when band densities were normalized to the density of actin at 42 kDa, there FIGURE 5. S-Nitrosylated proteins in cell lysates detected using the biotin-switch assay. Neutrophils or monocytes were obtained from wild type or MPO knock-out mice breathing just air (control) or after mice were exposed for 45 min to 2.8 ATA O 2 . As indicated, some wild type mice were used with no additional manipulations, whereas others were injected intraperitoneally with 40 mg/kg L-NAME 75 min before sacrifice (air-only exposure) or 30 min before the 45 min HBO 2 exposure. For identification of biotin-containing proteins such as ␤-actin, a degraded ␤-actin fraction, and profilin, bands were cut from nitrocellulose paper and subjected to MS as described under "Experimental Procedures." The 6 lanes on left side were contiguous on the same blot. The 2 lanes labeled as coming from KO mice were taken from a different blot. Numbers shown on the image are the increase in densities of bands relative to the densities of bands from control (air-exposure only) wild type neutrophil lysates run on the same blot. Numbers are mean Ϯ S.E. for 4 -13 independent trials. TX, treatment (L-NAME treatment). FIGURE 6. S-Nitrosylated proteins in cell lysates detected using the biotin-switch assay. Neutrophils were obtained from wild type mice breathing just air (control) or after mice were exposed for 45 min to 2.8 ATA O 2 . Cells were suspended in 1 ml of PBS containing 1 mM CaCl 2 , 1.5 mM MgCl 2 , and 5.5 mM glucose and incubated for 5 min at room temperature in the dark after addition of 10 l of PBS or 10 l of 10 M fMLP, or after addition of 10 l of PBS and incubation for 5 min under a UV light source. Cells were then centrifuged and cell lysates subjected to the biotin-switch assay. After Western blotting, bands were quantified, and the fold increase in the densities of the ␤-actin and profilin bands for the HBO 2 -exposed samples was compared against the ␤-actin or profilin band density from the air-exposed control cells incubated with just PBS that were run on the same Western blot. Data are means Ϯ S.E. for four trials. Densities of the bands from the HBO 2 -exposed cells incubated with fMLP or exposed to UV light were not significantly different from the densities for the control cell bands.
were no significant differences among the samples. We also probed blots for specific proteins to try and uncover what might be subtle alterations. We found no significant differences for bands recognized by an antibody to the CD18 component of ␤ 2 integrins, to proteins known to be involved with F-actin formation such as those related to the actin-related protein complex, including ArpC1A (41 kDa), ArpC3 (p21), mDia1(mammalian Diaphanous-related formin-1), and SPIRE1 (42)(43)(44) or a number of cytoskeletal proteins known to have altered associations with actin during cell activation such as talin, ␣-actinin, and profilin (45).
Cytoskeletal Analysis Based on Triton Solubility-The state of actin in neutrophils was also assessed by analyzing Tritonsoluble and -insoluble fractions of neutrophils plated onto fibrinogen (Fig. 11). When expressed as Triton-insoluble actin band density versus the sum of band densities on blots from Triton-soluble plus Triton-insoluble proteins, there was no significant difference in cytoskeletal F-actin content between air-only exposed, control neutrophils, and those obtained from mice exposed to HBO 2 . However, differences were clearly discerned between band densities for Triton-soluble F-actin and G-actin, which were separated by centrifuging Triton solutions at 336,000 ϫ g (see "Experimental Procedures"). There was significantly more Triton-soluble F-actin present in samples from HBO 2 -exposed neutrophils.
Western blots were also prepared to investigate whether there were differences in the Triton-insoluble concentration of a variety of proteins related to F-actin assembly, including cyclic GMP protein kinase, heat shock protein 20 (HSP20), phosphorylated HSP20, cofilin, and profilin. Among six separate trials, no differences were found when expressed as Triton-insoluble band density versus the sum of band densities on blots from Triton-soluble plus Triton-insoluble proteins (data not shown). However, we always noted that the band for profilin was denser in HBO 2 versus control on the Triton-insoluble blots. A subtle alteration in profilin content was detected when band densities for profilin and actin were analyzed on each Triton-insoluble blot. When normalized to the Tritoninsoluble actin band density present on the same blot, the profilin band was 1.41 Ϯ 0.06-fold (S.E., n ϭ 6, p Ͻ 0.05) more dense in blots from HBO 2 -exposed neutrophils versus blots from air-only exposed control neutrophils.
Ex Vivo Actin Polymerization and Network Formation-To further examine HBO 2 effects on actin, we evaluated the impact neutrophil lysates had on ex vivo actin polymerization. The polymerization rate of purified pyrene-labeled actin was markedly enhanced in the presence of neutrophil lysates from HBO 2 -exposed versus control air-exposed mice, and UV light abrogated this effect. Data in Table 3 show the initial linear rate of fluorescence increase. These findings are consistent with other studies showing that short actin filaments accelerate actin polymerization (46,47).
The apparent viscosity of polymerized actin solutions, thought to reflect actin network formation or gelation, was assessed in a low shear falling ball assay. The viscosity of actin solutions prepared with neutrophil lysates from HBO 2 -exposed mice was significantly higher than the viscosity of solutions prepared with lysates from control air-exposed mice. Higher viscosity of solutions was not observed using lysates prepared from neutrophils exposed to UV light (Table 3).

DISCUSSION
Results from this study demonstrate that neutrophils, but not monocytes, exhibit impaired ␤ 2 integrin adhesion after exposure to HBO 2 . Impaired adhesion occurs whether cells are  . Confocal microscope images of F-actin and CD18 (␤ 2 integrins) in permeabilized neutrophils. Neutrophils were obtained from mice breathing just air (control) or after mice were exposed for 45 min to 2.8 ATA O 2 . Cells were incubated at room temperature in the dark for 5 min and then placed onto fibrinogen-coated slides, incubated for 10 min, and then fixed with 2% paraformaldehyde before further processing as described under "Experimental Procedures." exposed to hyperoxia in vivo or isolated from animals and exposed ex vivo. From this we conclude that the effect of HBO 2 is intrinsic to the cells themselves and not based on generation of some intermediate in a systemic response that secondarily impacts neutrophils. HBO 2 -exposed cells liberate ⅐ NO. The HBO 2 effect was inhibited when mice were treated with L-NAME or 1400W, showing that iNOS activity is required for HBO 2 to impair ␤ 2 integrin-dependent adherence. MPO activity is also required, because HBO 2 inhibition did not occur in MPO KO mice. Absence of MPO is likely the explanation for why ␤ 2 integrins on monocytes are not inhibited by HBO 2 . Although peroxidase enzymes other than MPO can generate reactive nitrogen species, they are not as efficient as MPO (48). Nitrite, the major oxidation product of ⅐ NO, can react with hypochlorous acid generated by MPO to yield nitryl chloride (NO 2 Cl) and nitrogen dioxide (NO 2 ⅐ ) (49,50). Reactive nitrogen species such as NO 2 ⅐ can react with lipids, nucleic acid, and proteins. Under physiological conditions, the most rapid reactions are nitration of either of the two carbons in the phenol ring of tyrosine and S-nitrosylation of cysteine. The reaction with cysteine is approximately 2 orders of magnitude faster than that with tyrosine, so when reactions occur involving tyrosine-containing sulfhydryl proteins, NO 2 ⅐ primarily oxidizes solvent-exposed cysteine residues (51). We believe FIGURE 9. Fluorescence intensity of F-actin and CD18 (␤ 2 integrins) of permeabilized neutrophils. Neutrophils were obtained from mice breathing just air (control) or after mice were exposed for 45 min to 2.8 ATA O 2 . Cells were incubated at room temperature in the dark for 5 min Ϯ 100 nM fMLP, or under a UV light source for 5 min, and then placed onto fibrinogen-coated slides, incubated for 10 min, and then fixed with 2% paraformaldehyde before further processing as described under "Experimental Procedures." Data are mean Ϯ S.E. for a minimum of 100 individual cells obtained from at least four trials using different animals. *, p Ͻ 0.05. FIGURE 10. Western blot of actin immunoprecipitates. Neutrophils were obtained from mice breathing just air (control) or after mice were exposed for 45 min to 2.8 ATA O 2 . Cells were incubated for 5 min at room temperature in the dark, or under a UV light source, and then placed on fibrinogen-coated plates. After 10 min, proteins in adherent cells were cross-linked with DTSP, and cells were lysed and suspensions subjected to immunoprecipitation using anti-actin antibodies as described under "Experimental Procedures." The image shows a Western blot of the immunoprecipitated proteins, and this is representative of six separate trials. FIGURE 11. Western blots of neutrophil actin. Neutrophils were obtained from mice breathing just air (control) or after mice were exposed for 45 min to 2.8 ATA O 2 . Cells were placed on fibrinogen-coated plates for 10 min, and adherent cells were lysed with buffer containing Triton X-100 as described under "Experimental Procedures." A representative Western blot is shown as well as mean (ϮS.E., n ϭ 6) band densities for Triton-insoluble actin, actin removed by centrifuging the Triton solution (Triton-soluble F-actin), and actin remaining in the Triton after centrifugation (G-actin). *, p Ͻ 0.05. this explains why we did not find elevations of nitrotyrosine in proteins from HBO 2 -exposed neutrophils.
There were clear differences in the content of S-nitrosylated proteins in air-exposed control versus HBO 2 -exposed neutrophil lysates subjected to the biotin-switch assay. Amino acid sequencing of the bands suggest, surprisingly, that only actin and profilin are S-nitrosylated. Nitrosylated proteins were found in neutrophils from control mice, a reasonable finding given the presence of iNOS and MPO, but bands on Western blots were twice as dense in samples from HBO 2 -exposed cells. That bands were detected in the MPO-KO mice indicates that reactive species can be generated by peroxidase enzymes other than MPO.
The selectivity of protein S-nitrosylation reactions is under intense scrutiny, and the reasons why actin and possibly profilin were the only neutrophil proteins modified are unclear. Certain acid/base motifs and hydrophobic pockets have been shown to influence cysteine S-nitrosylation (52). Subcellular localization of enzymes that generate reactive nitrogen and oxygen species influence tyrosine nitration, and it would be reasonable that the same was also true for S-nitrosylation (53).
Neutrophil lysates were also treated with HPDP-biotin plus ascorbate and subjected to proteolysis, and then peptides with the HPDP-biotin moiety were affinity-captured. Sequences containing cysteine moieties with a mass shift because of HPDP-biotin were only obtained for actin. The biotin switch assay can yield false positive results because of disulfide adducts and detection of some oxidized thiols. The requisite control experiments suggested that S-nitrosylation of profilin occurs, i.e. no biotin-containing bands were identified on Western blots when proteins were incubated with HPDP-biotin or ascorbate, but not both, and with HgCl 2 , or if protein lysates were exposed to UV light. The basis for the discrepancy between the two approaches is not clear, and so the data do not provide unequivocal evidence of profilin modification.
Protein S-nitrosylation in response to HBO 2 exposure appears to be the basis for inhibition of ␤ 2 integrin function. This conclusion is supported by several lines of evidence. HBO 2 -mediated elevations of S-nitrosylated proteins did not occur in neutrophils from L-NAME-treated mice or in cells of MPO KO mice. Protein S-nitrosylation was also reversed by exposing cells to UV light. Under each of these circumstances, ␤ 2 integrin adherence was maintained despite HBO 2 exposure. S-Nitrosylation mediated by HBO 2 could also be reversed when cells were incubated with fMLP, and this treatment reversed the ␤ 2 integrin adhesion defect. fMLP activates neutrophils by binding to G-protein-coupled surface receptors and triggering a series of intracellular messengers, including phosphoinositides (28,29). This demonstrates that HBO 2 does not block the so-called inside-out pathway of neutrophil activation. In this regard, it is intriguing to consider that fMLP will activate cyclic GMP-dependent protein kinase and alter its intracellular localization in neutrophils (54,55). In other cell types, cyclic GMPdependent protein kinase regulates the expression of several proteins, including thioredoxin, which can de-nitrosylate (denitrosate) proteins (56 -58). We have reported that impaired ␤ 2 integrin function can be reversed by incubating HBO 2 -exposed cells with membrane-permeable 8-bromoguanosine 3Ј,5Ј-cyclic GMP (3,59). HBO 2 will inhibit the activity of neutrophil membrane guanylate cyclase but not the cytosolic form of the enzyme (3). Notably, in these prior studies cell adherence to multiple surfaces in addition to fibrinogen-coated plates was assayed, and fMLP did not reverse HBO 2 effects when cells interacted with some nonphysiological surfaces. The biochemical pathway leading to denitrosation of proteins will require further study.
HBO 2 clearly causes a degree of neutrophil activation based on membrane surface CD18 expression and oxidative burst. Imaging by confocal microscopy demonstrated that F-actin is markedly increased in neutrophils after HBO 2 exposure, and there is an absence of ␤ 2 integrin (CD18) clusters. The actin cytoskeleton is required for ␤ 2 integrin clustering, which increases receptor avidity (60,61). F-actin and CD18 clustering were normalized with UV light exposure and also by fMLP, linking these changes to cytoskeletal S-nitrosylation modifications triggered by HBO 2 . Absence of an inhibitory HBO 2 effect on ␤ 2 affinity as measured with ICAM-coated beads (Fig. 3) is consistent with observations that an intact actin cytoskeleton is not required for changes in ␤ 2 affinity (versus avidity or clustering) (60,61).
Nitrosylated cysteine moieties were found in the carboxylterminal area of actin, a region that is important for actin polymerization and for binding several proteins that modify behavior of the molecule (62). These proteins include profilin, myosin, cofilin, ␣-actinin, and gelsolin (63). Actin also plays a role in NADPH oxidase activation in neutrophils (64). When actin is incubated with S-nitrosoglutathione ex vivo, only the cysteine closest to the carboxyl-terminal end is S-nitrosylated. This chemical modification has no effect on G-actin polymerization of filament end-to-end annealing except under shear stress, where nitrosylated G-actin is found to polymerize less efficiently (65). Some modifications of the terminal cysteine moiety of actin can accelerate polymerization (possibly because the chemical modifications stabilize actin nuclei) and also slow cross-linking or actin network formation because of shorter filament length (66). Interestingly, nitration versus nitrosylation of G-actin reduces the critical monomer concentration from ϳ89 to Ͻ10 nM (67). Nitration seems to stabilize interactions between the pointed end of G-actin and the barbed ends of filaments that may stabilize formation of actin nuclei, accelerate filament elongation, and/or slow subunit dissociation. Actin nitration compromises several neutrophil functions (68). Nitrosylation of the four cysteine moieties closest to the carboxyl terminus clearly effects actin polymerization in several

Ex vivo G-actin polymerization rate and viscosity measurements
Neutrophils were isolated from air-breathing control mice or from mice first exposed to 2.8 ATA O 2 for 45 min; lysates were prepared, and 50 g of protein was used in each assay as described under "Experimental Procedures." Where indicated cells were exposed to UV light for 5 min and then lysed. Data are mean Ϯ S.E., n ϭ 5 for each sample.  (Table 3). It appears that the reason HBO 2 does not alter neutrophil functions such as degranulation (2, 3) and oxidative burst ( Table 2) is because concurrent with inside-out cell activation (e.g. fMLP exposure) actin nitrosylation is reversed. The F-actin content of HBO 2 -exposed neutrophils is markedly increased based on phallacidin binding, but cytoskeletal F-actin is not increased based on Triton insolubility. This discrepancy is reconciled by considering differences between these two approaches. Phallacidin binds large and small actin filaments but not G-actin (69). Triton is a traditional detergent used to assess F-actin and localization of proteins to the cytoskeleton (45,70). After cells are incubated with Triton and centrifuged, the supernatant contains G-actin and a cold-labile pool of short, noncross-linked F-actin, whereas the cell pellet contains cross-linked cytoskeletal F-actin. High speed centrifugation of the Triton solution will sediment the short F-actin filaments (39). HBO 2 increases the amount of short actin filaments in neutrophils, as assessed by phallacidin staining and measurements of Triton-soluble F-actin. Independent support for this conclusion comes from the effect cell lysates have on ex vivo polymerization of pyrene-labeled actin (Table 3), because a higher concentration of short actin filaments will accelerate actin polymerization (46,47). HBO 2 doubled the amount of Tritonsoluble F-actin, but based on phallacidin staining HBO 2 increased F-actin by nearly 3-fold (Figs. 9 and 11). We speculate that the apparent discrepancy in the amount of F-actin may relate to increased exposure of phallacidin-binding sites. Each phallacidin molecule binds to three neighboring actin subunits in a filament (71). Kinetic analysis indicates that at any one point in time relatively few phallacidin-binding sites are available on an actin filament (69). If S-nitrosylation of the four terminal cysteine moieties caused a more open filament structure (the term "breath" has been used in relation to actin filaments), this would explain the 3-fold fluorescence enhancement versus only a 2-fold increase in short F-actin filaments assessed by Triton solubility.
Based on Triton insolubility, HBO 2 exposure had a subtle effect on profilin association with the cytoskeleton. Variability in the band densities of Triton-soluble versus -insoluble profilin is likely to have caused our inability to detect the slight elevation associated with the cytoskeleton discerned by measuring profilin in the Triton-insoluble protein fraction and normalizing this to actin on the same blots. What impact this alteration may have on the cytoskeleton, or ␤ 2 integrin control, is not clear. Profilin participates in the microfilament system by interacting with actin, phosphoinositides, and poly(L-proline) (73). Under different conditions it may act to stabilize or sever filamentous actin, and profilin has both positive and negative effects on actin nucleation (74,75). Many proteins involved with regulating actin polymerization are known to recruit profilin to membrane structures (76,77). Because we did not observe differences in profilin cross-linked to actin by DTSP, we conclude that HBO 2 does not increase the amount of profilin directly associated with actin in neutrophils.
We did not detect HBO 2 -induced alterations of protein associations with actin based on DTSP cross-linking. This result leads us to conclude that changes intrinsic to actin are the basis for impaired ␤ 2 integrin adherence following exposure to HBO 2 . Affinity capture of ␤-actin after biotin switch indicates that ϳ66% of cellular actin is nitrosylated in HBO 2 -exposed neutrophils. As the proportion of nitrosylated actin added to G-actin solutions in the ex vivo studies was much less than 66%, these trials suggest that nitrosylated actin will perturb intracellular F-actin assembly. Actin polymerization is one mechanism that regulates integrin function (78).
It has been estimated that ␤-actin represents 13.2% of total protein in neutrophils (79). Our ex vivo studies were performed with 50 g of cell lysate protein, or about 6.6 g of actin, in solutions totaling 200 l. Hence, we used about 0.8 M neutrophil actin and ϳ0.53 M of the nitrosylated form of actin in studies with lysates from HBO 2 -exposed cells. Addition of HBO 2 -neutrophil lysate to 10 M G-actin (hence nitrosylated actin was ϳ5% of the total actin in the solution) significantly increased ex vivo pyrene-actin polymerization rate (that we hypothesize is because of the presence short F-actin filaments). Addition of lysate to 25 M G-actin (nitrosylated actin ϳ2% of the total actin in the solution) markedly increased the viscosity of polymerized actin in the ex vivo studies. This latter observation reflects gelation or formation of an actin filament network. Network formation is typically ascribed to filament cross-linking by a variety of actin-associated proteins; however, F-actin network stiffness can also be enhanced when actin filaments are at higher density because the filaments become entangled (80). We speculate that this entanglement causes the diffuse F-actin distribution within HBO 2 -exposed cells (Fig. 8). Because the confocal image of actin and also the elevation in ex vivo viscosity changes were abrogated by UV light exposure, we ascribe them to S-nitrosylated actin. This modification of the actin network is unique in that the rearrangement does not modify ␤-actin linkage to other cytoskeletal proteins inside neutrophils, and the F-actin remains Triton-soluble.
Our findings have clinical relevance because they improve understanding of HBO 2 effects on ␤ 2 integrins. As discussed in the Introduction, HBO 2 can ameliorate a variety of insults in animal and clinical trials. Inhibiting ␤ 2 integrins with monoclonal antibodies will also ameliorate ischemia-reperfusion injuries, but in contrast to HBO 2 , antibody therapy causes profound immunocompromise (81,82). Probably the most compelling evidence that HBO 2 does not cause immunocompromise comes from studies in sepsis models, where HBO 2 has a beneficial effect (83,84). We believe the molecular basis for why HBO 2 does not compromise neutrophil antibacterial functions is because hyperoxia does not block the inside-out pathway for activation (as shown by fMLP effects in this study) when actin S-nitrosylation is reversed.
There also may be physiological relevance to our observations. S-Nitrosylation events could be responsible for impairment of ␤ 2 integrin function observed in vivo and in vitro when neutrophils are exposed to a flux of ⅐ NO (85)(86)(87). We have previously reported that there are overlaps between the behavior of HBO 2 -exposed neutrophils and those exposed to ⅐ NO, although some differences were also noted (3,59,88).
In summary, reactive species derived from iNOS and MPO activity are responsible for excessive S-nitrosylation of ␤-actin, and possibly profilin, when neutrophils are subjected to hyper-oxia. HBO 2 causes S-nitrosylation of the four cysteine moieties closest to the carboxyl-terminal end of the molecule. This modification increases the concentration of short, noncross-linked F-actin filaments that form a cytoplasmic gelatin and inhibit ␤ 2 integrin clustering. We conclude that HBO 2 -mediated cytoskeletal changes inhibit ␤ 2 integrin-dependent neutrophil adhesion.
Among the questions raised by our observations, perhaps the most fundamental is the mechanism for HBO 2 -mediated production of reactive nitrogen species that perturb the cytoskeleton. MPO and iNOS co-localize within neutrophil primary granules (89). MPO-mediated nitrosylation reactions occur even when the rates of ⅐ NO and H 2 O 2 production are quite variable (50). Hyperoxia is expected to elevate intracellular H 2 O 2 concentration (25). By providing substrate for MPO, HBO 2 may enhance iNOS activity. Others have shown that MPO/H 2 O 2 up-regulates iNOS catalytic activity by scavenging free ⅐ NO and thereby decreasing the iNOS-nitrosyl complex (90). This pathway for iNOS modulation is rendered even more complex because ⅐ NO itself influences MPO activity. At low concentrations ⅐ NO can act as a ligand and substrate for MPO to enhance catalytic activity (by enhancing formation of compound II, the rate-limiting step) (91,92). Of course, dose-response issues will also influence this pathway because high ⅐ NO concentrations will react with MPO iron to limit catalytic activity (72). Further work is required to evaluate these issues.