Nitric Oxide Regulates Neutrophil Migration through Microparticle Formation
2007; Elsevier BV; Volume: 172; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.070069
ISSN1525-2191
AutoresSarah L. Nolan, Rachel Dixon, Keith E. Norman, Paul G. Hellewell, Victoria Ridger,
Tópico(s)Immune Response and Inflammation
ResumoThe role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation. The role of nitric oxide (NO) in regulating neutrophil migration has been investigated. Human neutrophil migration to interleukin (IL)-8 (1 nmol/L) was measured after a 1-hour incubation using a 96-well chemotaxis plate assay. The NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) significantly (P < 0.001) enhanced IL-8-induced migration by up to 45%. Anti-CD18 significantly (P < 0.001) inhibited both IL-8-induced and L-NAME enhanced migration. Antibodies to L-selectin or PSGL-1 had no effect on IL-8-induced migration but prevented the increased migration to IL-8 induced by L-NAME. L-NAME induced generation of neutrophil-derived microparticles that was significantly (P < 0.01) greater than untreated neutrophils or D-NAME. This microparticle formation was dependent on calpain activity and superoxide production. Only microparticles from L-NAME and not untreated or D-NAME-treated neutrophils induced a significant (P < 0.01) increase in IL-8-induced migration and transendothelial migration. Pretreatment of microparticles with antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) significantly (P < 0.001) inhibited this effect. The ability of L-NAME-induced microparticles to enhance migration was found to be dependent on the number of microparticles produced and not an increase in microparticle surface L-selectin or PSGL-1 expression. These data show that NO can modulate neutrophil migration by regulating microparticle formation. Accumulation and activation of inflammatory cells is vital to host defense but can also cause pathology. Neutrophils, for example, are critical for clearance of various pathogens but also cause injury and death of host tissue if their activity is misdirected or exaggerated.1Smith JA Neutrophils, host defense, and inflammation: a double-edged sword.J Leukoc Biol. 1994; 56: 672-686Crossref PubMed Scopus (773) Google Scholar Induction of inflammation is associated with increased expression or altered avidity of adhesion molecules on endothelial cells and leukocytes, which increases the likelihood of interaction between these cell types.2Lukacs NW Ward PA Inflammatory mediators, cytokines, and adhesion molecules in pulmonary inflammation and injury.Adv Immunol. 1996; 62: 257-304Crossref PubMed Google Scholar, 3Malik AB Lo SK Vascular endothelial adhesion molecules and tissue inflammation.Pharmacol Rev. 1996; 48: 213-229PubMed Google Scholar Initial attachment and rolling of neutrophils on endothelium is principally mediated by the selectin family of adhesion molecules, whereas stable adhesion and transmigration out of vessels is controlled by agents such as chemoattractants, integrins, members of the immunoglobulin superfamily, and junctional adhesion molecules.4Simon SI Green CE Molecular mechanics and dynamics of leukocyte recruitment during inflammation.Annu Rev Biomed Eng. 2005; 7: 151-185Crossref PubMed Scopus (227) Google Scholar, 5Kakkar AK Lefer DJ Leukocyte and endothelial adhesion molecule studies in knockout mice.Curr Opin Pharmacol. 2004; 4: 154-158Crossref PubMed Scopus (82) Google Scholar, 6Liu L Kubes P Molecular mechanisms of leukocyte recruitment: organ-specific mechanisms of action.Thromb Haemost. 2003; 89: 213-220PubMed Google Scholar, 7Rao RM Shaw SK Kim M Luscinskas FW Emerging topics in the regulation of leukocyte transendothelial migration.Microcirculation. 2005; 12: 83-89Crossref PubMed Scopus (17) Google Scholar, 8Nourshargh S Marelli-Berg FM Transmigration through venular walls: a key regulator of leukocyte phenotype and function.Trends Immunol. 2005; 26: 157-165Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar Nitric oxide (NO), a short-lived small molecule produced by most cell types, has a variety of well defined physiological and pathophysiological roles. A strong case for anti-inflammatory effects of NO is provided by studies showing that pharmacological inhibition of NO or genetic deletion of its synthases elevates leukocyte-endothelial cell interaction in diverse organs and tissues.9Akimitsu T Gute DC Korthuis RJ Leukocyte adhesion induced by inhibition of nitric oxide production in skeletal muscle.J Appl Physiol. 1995; 78: 1725-1732Crossref PubMed Scopus (46) Google Scholar, 10Hickey MJ Granger DN Kubes P Inducible nitric oxide synthase (iNOS) and regulation of leucocyte/endothelial cell interactions: studies in iNOS-deficient mice.Acta Physiol Scand. 2001; 173: 119-126Crossref PubMed Scopus (75) Google Scholar This case is supported by studies showing that NO-releasing compounds inhibit neutrophil migration.11Moilanen E Vuorinen P Kankaanranta H Metsa-Ketela T Vapaatalo H Inhibition by nitric oxide-donors of human polymorphonuclear leucocyte functions.Br J Pharmacol. 1993; 109: 852-858Crossref PubMed Scopus (147) Google Scholar, 12Benjamim CF Ferreira SH Cunha FQ Role of nitric oxide in the failure of neutrophil migration in sepsis.J Infect Dis. 2000; 182: 214-223Crossref PubMed Scopus (134) Google Scholar Early investigations using human umbilical vein endothelial cells suggested that NO regulates leukocyte recruitment by modulating adhesion molecule expression on endothelial cells,13De Caterina R Libby P Peng HB Thannickal VJ Rajavashisth TB Gimbrone Jr, MA Shin WS Liao JK Nitric oxide decreases cytokine-induced endothelial activation. 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Regulation of neutrophil function by NO is not straightforward however. In apparent conflict to the above, the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (L-NMMA) and the NO scavenger carboxy-PTIO attenuate neutrophil chemotaxis in vitro,16Wanikiat P Woodward DF Armstrong RA Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils.Br J Pharmacol. 1997; 122: 1135-1145Crossref PubMed Scopus (57) Google Scholar and exogenously generated NO has been reported to both induce17Beauvais F Michel L Dubertret L Exogenous nitric oxide elicits chemotaxis of neutrophils in vitro.J Cell Physiol. 1995; 165: 610-614Crossref PubMed Scopus (41) Google Scholar and inhibit16Wanikiat P Woodward DF Armstrong RA Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils.Br J Pharmacol. 1997; 122: 1135-1145Crossref PubMed Scopus (57) Google Scholar neutrophil chemotaxis, supporting both pro- and anti-inflammatory activities of this mediator. The complexities of the effects of NO on inflammatory responses were highlighted in a recent investigation using inducible (i)NOS-deficient mice.18Razavi HM Wang LF Weicker S Rohan M Law C McCormack DG Mehta S Pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase.Am J Respir Crit Care Med. 2004; 170: 227-233Crossref PubMed Google Scholar Neutrophil sequestration in the pulmonary microvasculature in response to cecal ligation and puncture was reduced in iNOS−/− mice compared with wild types. In contrast, accumulation of neutrophils in bronchoalveolar lavage fluid was enhanced in the iNOS−/− animals, suggesting that NO can have differential effects on different stages of leukocyte recruitment. Human neutrophils are reported to contain all three NOS enzymes.19de Frutos T Sanchez de Miguel L Farre J Gomez J Romero J Marcos-Alberca P Nunez A Rico L Lopez-Farre A Expression of an endothelial-type nitric oxide synthase isoform in human neutrophils: modification by tumor necrosis factor-alpha and during acute myocardial infarction.J Am Coll Cardiol. 2001; 37: 800-807Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Wallerath T Gath I Aulitzky WE Pollock JS Kleinert H Forstermann U Identification of the NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets.Thromb Haemost. 1997; 77: 163-167PubMed Google Scholar, 21Bryant Jr, JL Mehta P Von der Porten A Mehta JL Co-purification of 130 kD nitric oxide synthase and a 22 kD link protein from human neutrophils.Biochem Biophys Res Commun. 1992; 189: 558-564Crossref PubMed Scopus (35) Google Scholar, 22Evans TJ Buttery LD Carpenter A Springall DR Polak JM Cohen J Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria.Proc Natl Acad Sci USA. 1996; 93: 9553-9558Crossref PubMed Scopus (272) Google Scholar, 23Tsukahara Y Morisaki T Kojima M Uchiyama A Tanaka M iNOS expression by activated neutrophils from patients with sepsis.ANZ J Surg. 2001; 71: 15-20Crossref PubMed Scopus (28) Google Scholar However, it is generally accepted that only endothelial (e)NOS is constitutively expressed in unstimulated peripheral blood neutrophils. Depending on levels produced, NO may be released to influence nearby cells or may act as an intracellular messenger regulating the activity of the cell responsible for its generation. Inside cells, NO stimulates guanylyl cyclase to increase cGMP formation and may influence a number of factors including chemotaxis,24Kaplan SS Billiar T Curran RD Zdziarski UE Simmons RL Basford RE Inhibition of chemotaxis Ng-monomethyl-L-arginine: a role for cyclic GMP.Blood. 1989; 74: 1885-1887Crossref PubMed Google Scholar superoxide anion release,16Wanikiat P Woodward DF Armstrong RA Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils.Br J Pharmacol. 1997; 122: 1135-1145Crossref PubMed Scopus (57) Google Scholar integrin expression,25Conran N Gambero A Ferreira HH Antunes E de Nucci G Nitric oxide has a role in regulating VLA-4-integrin expression on the human neutrophil cell surface.Biochem Pharmacol. 2003; 66: 43-50Crossref PubMed Scopus (31) Google Scholar and F-actin polymerization.26Clancy R Leszczynska J Amin A Levartovsky D Abramson SB Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils.J Leukoc Biol. 1995; 58: 196-202PubMed Google Scholar These and other functional responses of neutrophils are highly polarized suggesting that the messenger systems supporting them are restricted to localized intracellular compartments. Discrepancies between experiments dealing with inhibition of endogenous NOS versus exogenous application of NO-generating compounds may be partially explained by localized versus widespread activity of NO. Given the complexity surrounding regulation of inflammation by NO, we used an in vitro transmigration assay to investigate direct effects of NO inhibition on human neutrophils. We find that the broad-spectrum NOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME) enhances neutrophil migration in response to the chemokine interleukin (IL)-8 via a mechanism that is dependent on adhesion between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1). We have also identified a mechanism for this enhancement, finding that L-NAME-treated neutrophils generate L-selectin- and PSGL-1-expressing microparticles and that these coat the surface of artificial migration chambers or endothelial cells to support enhanced migration of subsequently added neutrophils. Anti-human CD18 (6.5E) was a gift from M. Robinson, SLH Celltech Group, Slough, UK. Anti-human PSGL-1 (blocking PL-1 and nonblocking PL-2) were gifts from Professor R. McEver, University of Oklahoma, Norman, OK. Purified and phycoerythrin-conjugated anti-human L-selectin (DREG-200) and isotype control (MOPC-21) were purchased from Becton Dickinson (Oxford, UK). Venous blood was drawn from healthy adult volunteers and immediately transferred to tubes containing EDTA (1.6 mg/ml; Sarstedt Ltd., Beaumount Leys, Leicester, UK). Neutrophils were isolated from whole blood using a two-step density gradient. Briefly, 2.5 ml of high-density histopaque (1.119 g/ml; Sigma, Dorset, UK) was placed in a round-bottom 10-ml tube, and 2.5 ml of low-density histopaque (1.077 g/ml, Sigma) was carefully layered on top. Whole blood (5 ml) was then layered above the histopaque gradient and centrifuged for 30 minutes (700 × g at 20°C) to allow separation of the blood into its components. The granulocyte layer was harvested, resuspended in buffer [phosphate-buffered saline (PBS) containing 1 mmol/L Ca2+, 0.5 mmol/L Mg2, and supplemented with 0.1% low-endotoxin bovine serum albumin (BSA); Sigma], washed by centrifugation (350 × g for 6 minutes) and red blood cells lysed. Neutrophils were washed, counted using a hemocytometer, and centrifuged (350 × g for 6 minutes). Finally, neutrophils were diluted to the required concentration in RPMI (Invitrogen Ltd., Paisley, UK) supplemented with 0.1% BSA. Differential counts showed that preparations were consistently >97% neutrophils, which were >95% viable (as measured by Trypan blue dye exclusion). Neutrophil chemotaxis was measured in a 96-well chemotaxis chamber (Neuroprobe, Inc., Gaithersburg, MD) using a modification of the method described by Frevert and colleagues.27Frevert CW Wong VA Goodman RB Goodwin R Martin TR Rapid fluorescence-based measurement of neutrophil migration in vitro.J Immunol Methods. 1998; 213: 41-52Crossref PubMed Scopus (139) Google Scholar Wells were filled with 25 μl of human IL-8 (PeproTech, Rocky Hill, NJ), RPMI, or neutrophils (5 × 104) resuspended in RPMI. A filter membrane was positioned over the loaded wells, and 25 μl of neutrophils (2 × 106/ml) were placed directly onto 3.0-μm filter sites. The chamber was incubated for 1 hour (37°C in 5% CO2), and any nonmigrated neutrophils were removed from the upper surface of the filter by wiping and washing with 25-μl aliquots of RPMI. Both neutrophils that had migrated to the underside of the filter and those that had migrated into the lower wells were counted. To dislodge any migrated cells adherent to the underside of the filter membrane, the plate with the filter attached was centrifuged (350 × g for 10 minutes). The filter was removed, and neutrophils in the wells of the chemotaxis plate were resuspended and counted using a hemocytometer. To correct for chemokinesis, equal concentrations of chemoattractant were placed above and below the filter in control wells. To assess the role of adhesion molecules, neutrophils were resuspended in saturating doses of antibodies: anti-CD18 (6.5E, 6 μg/106), anti-L-selectin (DREG-200, 15 μg/106), anti-PSGL-1 (PL-1, 10 μg/106), or the appropriate control antibody. Aliquots (25 μl) were then deposited directly onto the filter membranes, and the transmigration assay performed as above. Human neutrophils (5 × 106/ml for each treatment, unless otherwise stated) were incubated with L-NAME (30 μmol/L; Tocris Cookson, Bristol, UK), its inactive enantiomer D-NAME (30 μmol/L, Sigma), N-formyl-Met-Leu-Phe (fMLP, 10 μmol/L; Sigma), or RPMI for 1 hour (37°C in 5% CO2) to stimulate microparticle formation. After incubation, microparticle-containing suspensions were cleared of large cellular fragments by centrifugation (350 × g for 6 minutes) and supernatants transferred to microcentrifuge tubes. After ultracentrifugation (100,000 × g for 2 hours),28Brodsky SV Zhang F Nasjletti A Goligorsky MS Endothelium-derived microparticles impair endothelial function in vitro.Am J Physiol. 2004; 286: H1910-H1915Google Scholar the microparticle pellets were resuspended in 100 μl of RPMI and 0.1% BSA. To investigate the effects of microparticles on neutrophil migration, neutrophils were incubated with L-NAME and the resulting microparticles isolated by centrifugation. This was followed by dialysis to remove any remaining L-NAME. Dialyzed microparticles (5-μl aliquots) were deposited onto the filter membranes of chemotaxis chambers and incubated for 30 minutes (37°C, 5% CO2) before addition of neutrophils and chemoattractants. Neutrophils were labeled with the membrane-intercalating red fluorescent dye PKH26 using a cell linker kit according to the manufacturer's instructions (Sigma). Microparticle formation was stimulated as above, and neutrophil suspensions, microparticle-containing supernatants, and filtered (0.2 μm) supernatants were compared using a FACScan flow cytometer (Becton Dickinson). Microparticles were defined as positive for red fluorescence and were identified and analytically separated from whole cells by their smaller forward and side scatter parameters and lower fluorescent intensities. A gate was positioned around the microparticle population and events analyzed for phosphatidylserine externalization using fluorescein isothiocyanate (FITC)-conjugated Annexin V (5 μl of Annexin 5 per 1 × 105 neutrophils; Becton Dickinson), which binds with high affinity to negatively charged phospholipids in the presence of calcium ions. The role of calpain and superoxide dismutase in microparticle generation was determined by adding the selective calpain inhibitor PD150606 or negative control PD145305 (1 to 3 μmol/L; Merck, Nottingham, UK)29Lokuta MA Nuzzi PA Huttenlocher A Calpain regulates neutrophil chemotaxis.Proc Natl Acad Sci USA. 2003; 100: 4006-4011Crossref PubMed Scopus (118) Google Scholar or superoxide dismutase (10 to 30 μg, Sigma) to isolated neutrophils together with the stimulus for microparticle formation. The number of microparticles formed was analyzed using flow cytometry as above. To determine adhesion molecule expression on the surface of microparticles, neutrophils were labeled with PKH26 as above and incubated with D-NAME (30 μmol/L) or L-NAME (30 μmol/L) for 1 hour (37°C, 5% CO2). The resulting microparticles were incubated for 30 minutes at 4°C with saturating concentrations of FITC-conjugated antibodies directed against L-selectin (DREG-200) and PSGL-1 (PL-1), with isotype control (mouse IgG1), or with binding nonblocking anti-PSGL-1 (PL-2). The effects of stimuli on neutrophil shape change were assessed using a method based on that described by Sabroe and colleagues.30Sabroe I Hartnell A Jopling LA Bel S Ponath PD Pease JE Collins PD Williams TJ Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways.J Immunol. 1999; 162: 2946-2955Crossref PubMed Google Scholar Rested isolated human neutrophils (5 × 105) were placed in 1.2-ml polypropylene cluster tubes and incubated with 100 μl of PBS, PMA (10−9 mol/L), fMLP (10−5 mol/L), D-NAME (3 × 10−5 mol/L), or L-NAME (3 × 10−5 mol/L) for 6 minutes in a shaking water bath set at 37°C. Tubes were then removed and 250 μl of ice-cold optimized fixative added to terminate the reaction. Effects on neutrophil shape change were analyzed immediately using flow cytometry as described by Sabroe and colleagues.30Sabroe I Hartnell A Jopling LA Bel S Ponath PD Pease JE Collins PD Williams TJ Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways.J Immunol. 1999; 162: 2946-2955Crossref PubMed Google Scholar Fluo 3-acetoxymethyl ester (Fluo 3-AM) was used to measure intracellular calcium mobilization in response to stimuli according to the technique described by Storey and colleagues.31Storey RF Sanderson HM White AE May JA Cameron KE Heptinstall S The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.Br J Haematol. 2000; 110: 925-934Crossref PubMed Scopus (272) Google Scholar Briefly, isolated neutrophils (5 × 106/ml) were resuspended in HEPES/Tyrode's buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 2.68 mmol/L KCl, 0.42 mmol/L Na2PO4, 11.9 mmol/L NaHCO3, 5 mmol/L glucose without CaCl2 and MgCl2), pH 7.4. To prevent leakage, probenecid (2.5 mmol/L) was added to all buffers.32Merritt JE McCarthy SA Davies MP Moores KE Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+.Biochem J. 1990; 269: 513-519Crossref PubMed Scopus (280) Google Scholar Neutrophils were incubated for 30 minutes (37°C, 5% CO2) with Fluo 3-AM (5 μmol/L, Invitrogen) and washed twice in buffer. Neutrophils were transferred to 1.2-ml polypropylene cluster tubes and CaCl2 (1 mmol/L) or EGTA (100 μmol/L) added. Baseline fluorescence was measured using flow cytometry. In selected samples, RPMI, D-NAME (30 μmol/L), L-NAME (30 μmol/L), or the calcium ionophore A23187 (10 μmol/L) was added, and fluorescence was immediately measured. The median fluorescence value (Ftest) was recorded every 15 seconds after stimulation for 2 minutes. Median fluorescence was also measured in an unlabeled sample (Fmin) and a sample containing A23187 (Fmax). Intracellular calcium concentration was calculated according to Storey and colleagues.31Storey RF Sanderson HM White AE May JA Cameron KE Heptinstall S The central role of the P(2T) receptor in amplification of human platelet activation, aggregation, secretion and procoagulant activity.Br J Haematol. 2000; 110: 925-934Crossref PubMed Scopus (272) Google Scholar Microparticle formation was induced as described above. Microparticle-containing pellets were fixed using 3% glutaraldehyde (Sigma) in 0.1 mol/L phosphate buffer. Secondary fixation was performed using 2% osmium tetroxide (Sigma). The samples were embedded in Araldite resin for 48 to 72 hours at 60°C. Ultra-thin sections (70 to 90 nm) were cut using a Reichert Ultracut E ultramicrotome (Reichert, Depew, NY). The sections were stained using 3% uranyl acetate in 50% ethanol (Sigma) followed by staining with Reynolds lead citrate for 25 minutes (Polysciences Inc., Eppelheim, Germany). The sections were analyzed under transmission electron microscopy (Philips CM10; Philips, Eindhoven, Holland), and micrographs were recorded on Kodak electron microscope film (Sigma, Dorset, UK). The methods used to investigate transendothelial migration were based on those described by Dunzendorfer and colleagues.33Dunzendorfer S Rothbucher D Schratzberger P Reinisch N Kahler CM Wiedermann CJ Mevalonate-dependent inhibition of transendothelial migration and chemotaxis of human peripheral blood neutrophils by pravastatin.Circ Res. 1997; 81: 963-969Crossref PubMed Scopus (103) Google Scholar Briefly, gelatin-coated Transwell filter inserts (Corning, Surrey, UK) were seeded with 1 × 105 human umbilical vein endothelial cells (HUVECs) and cultured in complete growth media in 24-well plates. Media were replaced every 2 days until a confluent monolayer was established (∼4 to 6 days after seeding). Monolayer permeability was checked using FITC-BSA (Sigma) leakage for 10, 30, 60, and 120 minutes. The monolayers were found to retain >93% FITC-BSA in the upper chamber of the plate. Once a confluent monolayer was established, neutrophil-derived microparticles were produced as described above. Media from the upper chamber was removed, the cells washed twice with PBS, and 20 μl of microparticles (derived from 5 × 106 neutrophils) were added to the filter. The plates were incubated for 30 minutes (37°C, 5% CO2) to allow the microparticles to coat the HUVECs. The Transwell filters were then moved to new plates and 600 μl of RPMI or IL-8 10−9 mol/L added to the lower wells. Neutrophils (5 × 105 in 100 μl of RPMI) were then added to the upper chamber and the plates incubated for 1 hour (37°C, 5% CO2) to allow migration to occur. The upper chambers were then carefully washed twice with fresh RPMI to remove any remaining neutrophils. To dislodge any migrated cells adherent to the underside of the filter membrane, the plate with the filter attached was centrifuged (350 × g for 10 minutes). The filter was removed and neutrophils in the wells of the chemotaxis plate were resuspended and counted using a hemocytometer. One set of wells was used to correct for chemokinesis by placing IL-8 (10−9 mol/L) in both the upper chambers and lower wells. Results are presented as mean ± SEM. Statistical analyses were performed using GraphPad Instat version 3.01 (GraphPad Software, San Diego, CA). One-way analysis of variance followed by Bonferroni's or Dunnett's test for multiple comparisons were performed on appropriate data sets. P values less than 0.05 were considered significant. IL-8 caused dose-dependent neutrophil migration that was maximal at 10−8 mol/L (Figure 1A). Vehicle (RPMI) did not induce significant neutrophil chemotaxis in this and subsequent experiments (chemokinesis control = 4.1 ± 0.8%, n = 3; RPMI = 7.4 ± 0.5%, n = 3). A submaximal concentration of 10−9 mol/L was chosen for subsequent experiments so that increases or decreases in IL-8-induced migration might be investigated. L-NAME (30 μmol/L) significantly enhanced IL-8-induced neutrophil migration, whereas the inactive enantiomer, D-NAME, did not (Figure 1B). Higher concentrations of L-NAME (100 μmol/L and 300 μmol/L) and D-NAME significantly reduced neutrophil viability (data not shown). The effect of L-NAME (30 μmol/L) on IL-8-induced migration was reversed by L-Arginine (10 mmol/L) but not the nonmetabolized enantiomer, D-Arginine (10 mmol/L, Figure 1B). These data suggest that enhanced neutrophil migration to IL-8 in the presence of L-NAME was a direct consequence of reduced NO production. Neutrophil migration toward IL-8 was significantly reduced by anti-CD18 (6.5E) antibody whether L-NAME was present or not (Figure 2A). The results demonstrate that CD18 is required for neutrophil migration toward IL-8, but do not identify the mechanism for enhanced migration induced by NO inhibition. Neutrophil-neutrophil and neutrophil-microparticle interactions, which may explain enhanced migration in the presence of L-NAME, may be L-selectin- and PSGL-1-dependent.34Bargatze RF Kurk S Butcher EC Jutila MA Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.J Exp Med. 1994; 180: 1785-1792Crossref PubMed Scopus (246) Google Scholar, 35Kunkel EJ Chomas JE Ley K Role of primary and secondary capture for leukocyte accumulation in vivo.Circ Res. 1998; 82: 30-38Crossref PubMed Scopus (81) Google Scholar, 36Sperandio M Smith ML Forlow SB Olson TS Xia L McEver RP Ley K P-selectin glycoprotein ligand-1 mediates l-selectin-dependent leukocyte rolling in venules.J Exp Med. 2003; 197: 1355-1363Crossref PubMed Scopus (206) Google Scholar We therefore investigated the role of L-selectin and PSGL-1 in enhanced IL-8-induced neutrophil migration in the presence of L-NAME (Figure 2, B and C). Interestingly, antibodies to L-selectin (DREG-200) or PSGL-1 (PL-1) had no effect on migration of untreated neutrophils to IL-8 but prevented the increased migration to IL-8 induced by L-NAME. Neutrophils incubated with L-NAME (Figure 3B) generated more microparticles than resting neutrophils (Figure 3A). The microparticles were analytically separated from intact neutrophils by their smaller forward (size) and side (granularity) scatter parameters and were positive for the membrane-binding dye PKH26GL. These microparticles were still present in large numbers after removal of intact neutrophils by centrifugation at 350 × g for 6 minutes (Figure 3C) but were removed by passage through a 0.2-μm filter (Figure 3D). The number of microparticles (as assessed by the number of events/minute counted in the microparticle gate) formed in response to L-NAME (3816 ± 370, n = 8) was comparable to that seen with the positive control, fMLP (3799 ± 288, n = 13), and was significantly (P < 0.05) different from vehic
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