Actin S-Nitrosylation Inhibits Neutrophil β2 Integrin Function
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m709200200
ISSN1083-351X
AutoresStephen R. Thom, Veena M. Bhopale, D. Joshua Mancini, Tatyana N. Milovanova,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoThe 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 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 β2 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 β1, β2, and β3 integrins, but the β2 family (known as CD18 integrins) is predominantly expressed, and β2 integrins play a central role in regulating neutrophil activation and endothelial adhesion (1Brown E. Lindberg F. Ann. Med. 1996; 28: 201-208Crossref PubMed Scopus (32) Google Scholar). When animals or humans are exposed to hyperbaric oxygen (HBO2) 3The abbreviations used are: HBO2, hyperbaric oxygen; fMLP, N-formyl-methionyl-leucine-phenylalanine; ATA, atmospheres absolute; biotin-HPDP, N-(6-(biotinamido)hexyl)-3′-(2′-pyridyldithio)propionamide; PBS, phosphate-buffered saline; NOS, nitric-oxide synthase; iNOS, inducible NOS; PMSF, phenylmethylsulfonyl fluoride; MPO, myeloperoxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTSP, dithiobis(succinimidyl propionate); ICAM, intercellular adhesion molecule; HBSS, Hanks' balanced salt solution; l-NAME, NG-nitro-l-arginine methyl ester; MS, mass spectrometry; DCF, 2′,7′-dichlorofluorescein; KO, knock-out; RPE, (R)-phycoerythrin; APC, allophycocyanin. 3The abbreviations used are: HBO2, hyperbaric oxygen; fMLP, N-formyl-methionyl-leucine-phenylalanine; ATA, atmospheres absolute; biotin-HPDP, N-(6-(biotinamido)hexyl)-3′-(2′-pyridyldithio)propionamide; PBS, phosphate-buffered saline; NOS, nitric-oxide synthase; iNOS, inducible NOS; PMSF, phenylmethylsulfonyl fluoride; MPO, myeloperoxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTSP, dithiobis(succinimidyl propionate); ICAM, intercellular adhesion molecule; HBSS, Hanks' balanced salt solution; l-NAME, NG-nitro-l-arginine methyl ester; MS, mass spectrometry; DCF, 2′,7′-dichlorofluorescein; KO, knock-out; RPE, (R)-phycoerythrin; APC, allophycocyanin. at 2.8-3.0 atmospheres absolute (ATA), the ability of circulating neutrophils to adhere to target tissues is temporarily inhibited (2Thom S.R. 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Surg. 2005; 130: 1623-1630Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Previous studies have shown that HBO2 does not reduce neutrophil viability or cause immunocompromise, and functions such as degranulation and oxidative burst in response to chemoattractants appear to be intact (2Thom S.R. Toxicol. Appl. Pharmacol. 1993; 123: 248-256Crossref PubMed Scopus (232) Google Scholar, 3Thom S.R. Mendiguren I. Hardy K. Bolotin T. Fisher D. Nebolon M. Kilpatrick L. Am. J. Physiol. 1997; 272: C770-C777Crossref PubMed Google Scholar). 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 (20Cambi A. Joosten B. Koopman M. Lange F.D. Beeren I. Torensma R. Fransen J. Garcia-Parajo M. Leeuwen F.V. Figdor C. Mol. Biol. Cell. 2006; 17: 4270-4281Crossref PubMed Scopus (101) Google Scholar, 21Hato T. Pampori N. Shattil S. J. Cell Biol. 1998; 141: 1685-1695Crossref PubMed Scopus (211) Google Scholar). 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 (22Calderwood D. Shattil S. Ginsberg M. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 23Blystone S. Biochim. Biophys. Acta. 2004; 1692: 47-54Crossref PubMed Scopus (42) Google Scholar). There are over 50 proteins involved with connecting integrins and the actin cytoskeleton (24Zamir E. Geiger B. J. Cell Sci. 2001; 114: 3583-3590Crossref PubMed Google Scholar). The goal of this investigation was to elucidate the mechanism for the inhibitory effect of HBO2 on neutrophil β2 integrin function. We hypothesized that the HBO2 mechanism was related to production of reactive species. Production of superoxide radical ( (O2·)) and hydrogen peroxide (H2O2) is increased by hyperoxia, and HBO2 can also increase nitric oxide (.NO) production by stimulating isoforms of nitric-oxide synthase (NOS) (25Jamieson D. Chance B. Cadenas E. Boveris A. Annu. Rev. Physiol. 1986; 48: 703-719Crossref PubMed Scopus (316) Google Scholar, 26Thom S.R. Bhopale V. Fisher D. Manevich Y. Huang P.L. Buerk D.G. J. Neurobiol. 2002; 51: 85-100Crossref PubMed Scopus (78) Google Scholar, 27Thom S.R. Fisher D. Zhang J. Bhopale V.M. Ohnishi S.T. Kotake Y. Ohnishi T. Buerk D.G. Am. J. Physiol. 2003; 284: H1230-H1239Crossref PubMed Scopus (89) Google Scholar). In this study we show that neutrophil NOS and myeloperoxidase (MPO) are required for β2 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 inside-out path, can override the HBO2 effect (28Sha'afi R. Molski T. Prog. Allergy. 1988; 42: 1-64PubMed Google Scholar, 29Zaffran Y. Lepidi H. Bongrand P. Mege J.-L. Capo C. J. Cell Sci. 1993; 105: 675-684Crossref PubMed Google Scholar). Ultraviolet light (UV) photo-reversal of protein S-nitrosylation also restores β2 integrin function. Thus, HBO2 inhibits neutrophil β2 integrin activation by generating reactive nitrogen species that perturb the cytoskeleton. Materials—Unless otherwise noted, chemicals were purchased from Sigma. N-(6-(Biotinamido)hexyl)-3′-(2′-pyridyldithio)propionamide (biotin-HPDP) and streptavidin-Sepharose were purchased from Pierce. Ultrafree-MC filters, polyvinylidene difluoride Immobilon-FL, and ZipTipC18P10 were from Millipore Corp. Antibodies were purchased from the following vendors: anti-biotin, anti-talin, and anti-actin from Sigma; anti-cGMP protein kinase, anti-HSP20, anti-phospho-HSP20, anti-cofilin, anti-profilin, anti-α-actinin, and goat anti-mouse IgG conjugated to APC from BD Biosciences; anti-CD66, APC-conjugated and fluorescein isothiocyanate-conjugated anti-Ly-6G from Cell Sciences (Canton, MA); anti-mouse CD18 and CD18-RPE conjugate from eBiosciences (San Diego, CA); anti-human Fc receptor mouse IgG from Jackson ImmunoResearch (West Grove, PA); intercellular adhesion molecule (ICAM)/human IgG chimera fromR&D Systems (Minneapolis, MN); and anti-ArpC1A, anti-ArpC3, anti-mDia1(mammalian Diaphanous-related formin-1), and anti-SPIRE1 from Novus Biologicals (Littleton, CO). Animals—Wild type mice (Mus musculus) and MPO knock-out 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 (Indian-apolis, IN). Mice and rats were exposed to O2 at 1-3 ATA for 45 min following our published protocol (30Thom S.R. Bhopale V.M. Velazquez O.C. Goldstein L.J. Thom L.H. Buerk D.G. Am. J. Physiol. 2006; 290: H1378-H1386Crossref PubMed Scopus (232) Google Scholar). 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. (31Fekete Z. Hauser C.J. Adams Jr., J.M. Adams Jr., C.A. Forsythe R.M. Haskó G. Xu D.Z. Livingston D.H. Deitch E.A. Shock. 2001; 16: 15-20Crossref PubMed Scopus (20) Google Scholar). Blood was made to a volume of 2.5 ml with cell buffer (PBS containing 1 mm CaCl2, 1.5 mm MgCl2, 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 × 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 × g for 10 min. The supernatant was discarded, and cell pellets were resuspended in NH4Cl/Tris Lysis Buffer for 10 min. The solutions were centrifuged at 450 × g for 10 min, and the cells were resuspended in cell buffer at a concentration of 5 × 106/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 H2SO4 and rinsing three times with distilled water. Fibrinogen (500 μl of solution containing 30 μg/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 HBO2, were incubated for 30 min at 37 °C with calcein AM (5 μg/ml), washed twice with cell buffer, and then resuspended to a count of 5 × 105/100 μl. Aliquots of 100 μl were incubated on plates either alone, with anti-CD18 antibody (4 μg/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 μl 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 β2 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 concentration-dependent and was determined empirically. Compensation for multicolor assays was performed automatically by FACSDiva software on the LSRII. Identification and enumeration of ICAM beads, β-integrins, and neutrophils were performed using CellQuest™ acquisition/analysis software (BD Biosciences), including three-dimensional histograms for Quantum Plex™ SP microbeads quantification and the FlowJo Program (Tree-Star Inc., Ashland, OR). Surface Expression of β2 Integrins—After mice were exposed to HBO2, neutrophils were obtained and incubated with 4 μl 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 × 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 neutrophil gating location was verified by co-labeling cells with anti-CD66 or anti-Ly-6G (32Ruchaud-Sparagano M. Stocks S. Turley H. Dransfield I. Br. J. Haematol. 1997; 98: 612-620Crossref PubMed Scopus (20) Google Scholar, 33Zhou J. Stohlman S. Hinton B. Marten N. J. Immunol. 2003; 170: 3331-3336Crossref PubMed Scopus (119) Google Scholar). These extra manipulations did not modify β2 integrin expression. Oxidative Burst—After isolation, neutrophils were incubated with 5 μm 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 × 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™ SP beads (Bangs Laboratories, Fishers, IN; 50 μl, ∼1 × 105 red-stained 4.4 μm beads that fluoresce in FL3 channel) with 50 μl of anti-human Fc receptor mouse IgG and incubated for 30 min at room temperature. Suspensions were centrifuged at 1000 × g for 10 min, washed twice, and resuspended in 100 μl of PBS. Then 25 μg of mouse ICAM-1/human Fc chimera was added and incubated for 1 h at room temperature, followed by 20 μl 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 μl of PBS, and stored at 4 °C until use. Isolated neutrophils from control or HBO2-exposed mice were counted and incubated with beads at a ratio of ∼20 beads/cell in the absence or presence of 100 nm fMLP. Adhesion was determined by flow cytometry. Microelectrode Assay—Production of .NO by cells exposed to various partial pressures of O2 was carried out using published procedures (26Thom S.R. Bhopale V. Fisher D. Manevich Y. Huang P.L. Buerk D.G. J. Neurobiol. 2002; 51: 85-100Crossref PubMed Scopus (78) Google Scholar, 34Thom S.R. Xu Y.A. Ischiropoulos H. Chem. Res. Toxicol. 1997; 10: 1023-1031Crossref PubMed Scopus (145) Google Scholar). In brief, neutrophils were suspended in cell buffer at a concentration of 2.5 × 106/ml and placed in a hyperbaric chamber with pass-through connections where a polarographic .NO meter (ISO-NOP200, World Precision Instruments, Sarasota, FL.) had been mounted and calibrated using authentic .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 O2 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 O2, the difference in responses was compared. The study was then repeated, but cells were suspended in buffer containing 1 mm NG-nitro-l-arginine methyl ester (l-NAME). The difference in signals ±1 mml-NAME was taken as .NO production by the cells. Cell Extract Preparation and Biotin-switch Assay—Neutrophils were isolated from control and HBO2-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 × 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. (35Jaffrey S. Erdjument-Bromage H. Ferris C. Tempst P. Snyder S. Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1222) Google Scholar). After adding 4 volumes of blocking buffer (HEN buffer + 2.5% SDS, 20 mm methyl methanethiosulfonate), samples were incubated at 50 °C for 20 min with frequent vortexing. Ten volumes of -20 °C acetone was added and incubated at -20 °C for 20 min, followed by centrifugation at 2000 × g for 10 min and resuspension in 0.1 ml of HENS buffer + 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, biotin-switch analysis was performed on cell lysates treated with HPDP-biotin or with ascorbate (but not both) or with 1 mm HgCl2 for 10 min at room temperature (which cleaves S-NO bonds). For initial identification of nitrosylated proteins, 2 volumes of -20 °C acetone were added to lysate samples and incubated at -20 °C for 20 min, and samples were centrifuged at 2000 × 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 (36Thom S.R. Bhopale V.M. Fisher D. Zhang J. Gimotty P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13660-13665Crossref PubMed Scopus (208) Google Scholar). 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 μl of dry, washed streptavidin-Sepharose beads for 30 min. After centrifugation at 5,000 × 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 × g for 4 min, the supernatant was removed, and peptide fragments were eluted with 70% formic acid and evaporated to ∼5 μl in vacuo, followed by resuspension in 20 μl 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 nanoliquid chromatography/nanospray/LTQ mass spectrometer following our published methods (36Thom S.R. Bhopale V.M. Fisher D. Zhang J. Gimotty P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13660-13665Crossref PubMed Scopus (208) Google Scholar). 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 μl of 0.1% formic acid + 1% acetonitrile and 3 μl 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 HPDP-biotin in the y- or b-ion series were considered. The cross-correlation (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 HBO2-exposed mice were placed on glass slides that had been coated with fibrinogen. Poly-l-lysine-coated slides were incubated with a solution of fibrinogen (200 μg/ml) for 3 h at 37 °C and then cooled to room temperature prior to use. An 80-μl cell suspension was incubated on slides for 10 min and then fixed by addition of 80 μl 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 NH4Cl for 15 min and then rinsed with PBS. Cells were then permeabilized by adding 2 mm palmitoyl l-α-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 HBO2-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. (37Huttelmaier S. Mayboroda O. Harbeck B. Jarchau T. Jockusch B. Rudiger M. Curr. Biol. 1998; 8: 479-488Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) using dithiobis(succinimidyl propionate) (DTSP) (Pierce). DTSP cross-links proteins over a span of ∼12 Å (37Huttelmaier S. Mayboroda O. Harbeck B. Jarchau T. Jockusch B. Rudiger M. Curr. Biol. 1998; 8: 479-488Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). After 300 μl 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 + 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 μg/ml leupeptin, 0.5 μg/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 × g for 10 min, and the supernatant was collected. A pre-cleared supernatant volume containing 250 μg of protein was incubated with 5 μg of anti-actin antibodies on a shaker overnight at 4 °C, and then 30 μl 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 μl of heated SDS buffer (62.5 mm Tris-HCl, 2% SDS, 10% glycerol, 20% β-mercaptoethanol), and incubated at 95 °C for 15-20 min. Proteins were subjected to electrophoresis in 12% SDS-polyacrylamide gels, and Western blot analysis was performed as described previously (36Thom S.R. Bhopale V.M. Fisher D. Zhang J. Gimotty P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13660-13665Crossref PubMed Scopus (208) Google Scholar). Cytoskeletal Protein Analysis Based on Triton Solubility—Neutrophil suspensions from control or HBO2-exposed mice were
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