CD40 Ligand Promotes Mac-1 Expression, Leukocyte Recruitment, and Neointima Formation after Vascular Injury
2008; Elsevier BV; Volume: 172; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2008.070633
ISSN1525-2191
AutoresGuohong Li, John M. Sanders, Melissa Bevard, ZhiQi Sun, James W. Chumley, Elena Galkina, Klaus Ley, Ian J. Sarembock,
Tópico(s)Coronary Interventions and Diagnostics
ResumoHigh levels of circulating soluble CD40 ligand (sCD40L) are frequently found in patients with hypercholesterolemia, diabetes, ischemic stroke, or acute coronary syndromes, predicting an increased rate of atherosclerotic plaque rupture and restenosis after coronary/carotid interventions. Clinical restenosis is characterized in part by exaggerated neointima formation, but the underlying mechanism remains incompletely understood. This study investigated the role of elevated sCD40L in neointima formation in response to vascular injury in an atherogenic animal model and explored the molecular mechanisms involved. apoE−/− mice fed a Western diet developed severe hypercholesterolemia, significant hyperglycemia, and high levels of plasma sCD40L. Neointima formation after carotid denudation injury was exaggerated in the apoE−/− mice. In vivo, blocking CD40L with anti-CD40L monoclonal antibody attenuated the early accumulation of Ly-6G+ neutrophils and Gr-1+ monocytes (at 3 days) and the late accumulation of Mac-2+ macrophages (at 28 days) in the denudated arteries; it also reduced the exaggerated neointima formation at 28 days. In vitro, recombinant CD40L stimulated platelet P-selectin and neutrophil Mac-1 expression and platelet-neutrophil co-aggregation and adhesive interaction. These effects were abrogated by anti-CD40L or anti-Mac-1 monoclonal antibody. Moreover, recombinant CD40L stimulated neutrophil oxidative burst and release of matrix metalloproteinase-9 in vitro. We conclude that elevated sCD40L promotes platelet-leukocyte activation and recruitment and neointima formation after arterial injury, potentially through enhancement of platelet P-selectin and leukocyte Mac-1 expression and oxidative activity. High levels of circulating soluble CD40 ligand (sCD40L) are frequently found in patients with hypercholesterolemia, diabetes, ischemic stroke, or acute coronary syndromes, predicting an increased rate of atherosclerotic plaque rupture and restenosis after coronary/carotid interventions. Clinical restenosis is characterized in part by exaggerated neointima formation, but the underlying mechanism remains incompletely understood. This study investigated the role of elevated sCD40L in neointima formation in response to vascular injury in an atherogenic animal model and explored the molecular mechanisms involved. apoE−/− mice fed a Western diet developed severe hypercholesterolemia, significant hyperglycemia, and high levels of plasma sCD40L. Neointima formation after carotid denudation injury was exaggerated in the apoE−/− mice. In vivo, blocking CD40L with anti-CD40L monoclonal antibody attenuated the early accumulation of Ly-6G+ neutrophils and Gr-1+ monocytes (at 3 days) and the late accumulation of Mac-2+ macrophages (at 28 days) in the denudated arteries; it also reduced the exaggerated neointima formation at 28 days. In vitro, recombinant CD40L stimulated platelet P-selectin and neutrophil Mac-1 expression and platelet-neutrophil co-aggregation and adhesive interaction. These effects were abrogated by anti-CD40L or anti-Mac-1 monoclonal antibody. Moreover, recombinant CD40L stimulated neutrophil oxidative burst and release of matrix metalloproteinase-9 in vitro. We conclude that elevated sCD40L promotes platelet-leukocyte activation and recruitment and neointima formation after arterial injury, potentially through enhancement of platelet P-selectin and leukocyte Mac-1 expression and oxidative activity. 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L'Allier et al16L'Allier PL Tardif JC Gregoire J Joyal M Lesperance J Fortier A Guertin MC Sustained elevation of serum CD40 ligand levels one month after coronary angioplasty predicts angiographic restenosis.Can J Cardiol. 2005; 21: 495-500PubMed Google Scholar reported that elevation of plasma sCD40L persisted for at least 1 month after angioplasty and correlated with angiographic late lumen loss at 6-month follow-up. Although these clinical observations are suggestive of a causal relationship, whether and how elevated plasma sCD40L contributes to neointima formation after arterial injury have not been investigated.Because most patients who undergo PCI have underlying atherosclerosis and also frequently have hypercholesterolemia or diabetes, studies of the mechanisms of neointima formation and restenosis in preclinical models should attempt to mimic clinically relevant conditions. We and others have previously reported that atherosclerosis-prone apoE-deficient (apoE−/−) mice develop severe hypercholesterolemia, accelerated atherosclerosis, significant insulin resistance, and hyperglycemia when fed an atherogenic Western diet (WD).17Phillips JW Barringhaus KG Sanders JM Yang Z Chen M Hesselbacher S Czarnik AC Ley K Nadler J Sarembock IJ Rosiglitazone reduces the accelerated neointima formation after arterial injury in a mouse injury model of type 2 diabetes.Circulation. 2003; 108: 1994-1999Crossref PubMed Scopus (71) Google Scholar, 18Su Z Li Y James JC Matsumoto AH Helm GA Lusis AJ Shi W Genetic linkage of hyperglycemia, body weight and serum amyloid-P in an intercross between C57BL/6 and C3H apolipoprotein E-deficient mice.Hum Mol Genet. 2006; 15: 1650-1658Crossref PubMed Scopus (31) Google Scholar, 19Hansmann G Wagner RA Schellong S Perez VA Urashima T Wang L Sheikh AY Suen RS Stewart DJ Rabinovitch M Pulmonary arterial hypertension is linked to insulin resistance and reversed by peroxisome proliferator-activated receptor-gamma activation.Circulation. 2007; 115: 1275-1284Crossref PubMed Google Scholar The present study further showed that WD-fed apoE−/− mice developed high levels of plasma sCD40L as well as exaggerated neointima formation after carotid wire denudation injury compared with chow-fed apoE−/− mice. Therefore, this model provides a unique opportunity for understanding the role of elevated circulating CD40L in neointima formation after vascular injury under clinically relevant conditions.Leukocyte recruitment plays an essential role in the mechanism of neointima formation and restenosis.20Shah PK Inflammation, neointimal hyperplasia, and restenosis: as the leukocytes roll, the arteries thicken.Circulation. 2003; 107: 2175-2177Crossref PubMed Scopus (103) Google Scholar, 21Welt FG Rogers C Inflammation and restenosis in the stent era.Arterioscler Thromb Vasc Biol. 2002; 22: 1769-1776Crossref PubMed Scopus (536) Google Scholar Mac-1 (CD11b/CD18, αMβ2) is the predominant leukocyte-specific β2 integrin. It is up-regulated on activated leukocytes and binds to multiple ligands or counter-receptors such as fibrinogen, platelet glycoprotein Ibα (GPIbα), and intercellular adhesion molecule-1 (ICAM-1) and ICAM-2.22Weber C Springer TA Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to alphaIIbbeta3 and stimulated by platelet-activating factor.J Clin Invest. 1997; 100: 2085-2093Crossref PubMed Scopus (312) Google Scholar, 23Simon DI Chen Z Xu H Li CQ Dong J McIntire LV Ballantyne CM Zhang L Furman MI Berndt MC Lopez JA Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18).J Exp Med. 2000; 192: 193-204Crossref PubMed Scopus (499) Google Scholar, 24Wang Y Sakuma M Chen Z Ustinov V Shi C Croce K Zago AC Lopez J Andre P Plow E Simon DI Leukocyte engagement of platelet glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological response to vascular injury.Circulation. 2005; 112: 2993-3000PubMed Google Scholar Mac-1 has been shown to be a crucial regulator of leukocyte recruitment by promoting leukocyte firm adhesion to adherent platelets and fibrinogen at the sites of injured arteries, and has been associated with neointimal hyperplasia after vascular injury.24Wang Y Sakuma M Chen Z Ustinov V Shi C Croce K Zago AC Lopez J Andre P Plow E Simon DI Leukocyte engagement of platelet glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological response to vascular injury.Circulation. 2005; 112: 2993-3000PubMed Google Scholar Clinical studies demonstrate that activation of circulating neutrophils (as determined by enhanced Mac-1 expression) is associated with late lumen loss and restenosis.25Neumann FJ Ott I Gawaz M Puchner G Schomig A Neutrophil and platelet activation at balloon-injured coronary artery plaque in patients undergoing angioplasty.J Am Coll Cardiol. 1996; 27: 819-824Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 26Inoue T Uchida T Yaguchi I Sakai Y Takayanagi K Morooka S Stent-induced expression and activation of the leukocyte integrin Mac-1 is associated with neointimal thickening and restenosis.Circulation. 2003; 107: 1757-1763Crossref PubMed Scopus (110) Google Scholar, 27Inoue T Kato T Hikichi Y Hashimoto S Hirase T Morooka T Imoto Y Takeda Y Sendo F Node K Stent-induced neutrophil activation is associated with an oxidative burst in the inflammatory process, leading to neointimal thickening.Thromb Haemost. 2006; 95: 43-48PubMed Google Scholar, 28Inoue T Sakai Y Hoshi K Yaguchi I Fujito T Morooka S Lower expression of neutrophil adhesion molecule indicates less vessel wall injury and might explain lower restenosis rate after cutting balloon angioplasty.Circulation. 1998; 97: 2511-2518Crossref PubMed Scopus (126) Google Scholar, 29Inoue T Sakai Y Morooka S Hayashi T Takayanagi K Takabatake Y Expression of polymorphonuclear leukocyte adhesion molecules and its clinical significance in patients treated with percutaneous transluminal coronary angioplasty.J Am Coll Cardiol. 1996; 28: 1127-1233Abstract Full Text PDF PubMed Scopus (101) Google Scholar However, the mechanism underlying neutrophil activation and recruitment in the response to vascular injury is largely unknown. The present study investigated the role of elevated sCD40L in leukocyte recruitment and neointimal formation in response to vascular injury in an atherogenic animal model, and explored the molecular mechanisms involved.Materials and MethodsReagents and AntibodiesRecombinant mouse and human soluble CD40 ligand (rm-CD40L, rh-CD40L) was purchased from R&D Systems Inc. (Minneapolis, MN). Other reagents were from Sigma Chemical Co. (St. Louis, MO), unless otherwise specified. Function-blocking anti-human CD40L (TRAP1) and anti-human CD11b/Mac-1 (ICRF44) monoclonal antibodies (mAbs) were purchased from BD Biosciences (San Jose, CA). Function-blocking anti-mouse CD40 (HM40–3) and anti-mouse CD40L (MR1) mAbs were purchased from eBiosciences Inc. (San Diego, CA) and Taconic Biotechnology (Germantown, MD), respectively. Function-blocking anti-mouse integrin CD11b mAb (M1/70, αM chain of Mac-1) was from eBioscience Inc. Normal Armenian hamster IgG was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Fluorescence-conjugated antibodies were from BD Biosciences PharMingen (San Diego, CA), unless otherwise specified.Mouse Injury Model and Antibody TreatmentMale C57/BL6 apoE−/− mice 8 to 10 weeks of age (18–20 g; stock no. 002052, The Jackson Laboratory, Bar Harbor, ME) were used for these experiments. Mice were fed with a Western-type diet (TD 88137; Harlan-Teklad, Madison, WI) containing 21% fat by weight (0.15% cholesterol and 19.5% casein without sodium cholate) for 1 week before and 4 weeks after carotid wire injury, as previously described.17Phillips JW Barringhaus KG Sanders JM Yang Z Chen M Hesselbacher S Czarnik AC Ley K Nadler J Sarembock IJ Rosiglitazone reduces the accelerated neointima formation after arterial injury in a mouse injury model of type 2 diabetes.Circulation. 2003; 108: 1994-1999Crossref PubMed Scopus (71) Google Scholar, 30Li G Sanders JM Phan ET Ley K Sarembock IJ Arterial macrophages and regenerating endothelial cells express P-selectin in atherosclerosis-prone apolipoprotein E-deficient mice.Am J Pathol. 2005; 167: 1511-1518Abstract Full Text Full Text PDF PubMed Scopus (40) Google ScholarThe mouse carotid artery wire injury model of Lindner et al31Lindner V Fingerle J Reidy MA Mouse model of arterial injury.Circ Res. 1993; 73: 792-796Crossref PubMed Scopus (305) Google Scholar was used with minor modification, as previously published.17Phillips JW Barringhaus KG Sanders JM Yang Z Chen M Hesselbacher S Czarnik AC Ley K Nadler J Sarembock IJ Rosiglitazone reduces the accelerated neointima formation after arterial injury in a mouse injury model of type 2 diabetes.Circulation. 2003; 108: 1994-1999Crossref PubMed Scopus (71) Google Scholar, 30Li G Sanders JM Phan ET Ley K Sarembock IJ Arterial macrophages and regenerating endothelial cells express P-selectin in atherosclerosis-prone apolipoprotein E-deficient mice.Am J Pathol. 2005; 167: 1511-1518Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Animals were handled in compliance with the Guiding Principles in the Care and Use of Animals. Protocol approval was obtained from the Animal Research Committee of the University of Virginia Health System.In the in vivo study, mice were randomly divided into three experimental groups: 1) MR1 mAb-treated group, 2) (hamster IgG) isotype control-treated group, and 3) no antibody treatment. Starting 3 hours before carotid injury, each mouse was given 250 μg of MR1 mAb or isotype control Ab every 3 days until sacrifice via intraperitoneal injection with the operator blinded to treatment.Morphometric Analysis and ImmunohistochemistryThe arterial segments were dehydrated in ethanol and xylene and embedded in paraffin. Sections (5 μm thick) were stained by the Movat method.32Movat H Demonstration of all connective tissue elements in a single section.Arch Pathol Med. 1955; 60: 289-295Google Scholar Histomorphometric analysis was performed by individuals blinded to treatment group. For quantitative histopathological comparisons, the mean data of 10 sections was taken. The area of the lumen, internal elastic lamina, and external elastic lamina were determined by planimetry using Image Pro Plus 3.0 (Media Cybernetics), and the lumen area, plaque area, medial area, intima to media ratio, and overall vessel area were calculated. Neointima area was calculated by subtracting lumen area from the internal elastic lamina area, and medial area was determined by subtracting the internal elastic lamina area from the external elastic lamina area. Arterial size was measured by tracing the circumference of the external elastic lamina.Paraffin-embedded arterial sections were processed for immunohistochemistry with the avidin-biotin-peroxidase method (Vector Laboratories) using smooth muscle α-actin mAb (α-SMA; Sigma Chemical Co.) to detect vascular smooth muscle cells (SMCs); Mac-2 (M3/38, Accurate) was used to detect macrophages. For quantitative comparisons of macrophage or smooth muscle cell content, sections were digitized and the number of positively stained pixels were counted and normalized to total neointima and medial area using Image Pro Plus 3.0 (Media Cybernetics).Flow Cytometry Analysis of Leukocytes Accumulated in Mouse Carotid ArteriesFlow cytometry was performed to assess the total leukocyte content and the subpopulations of leukocytes accumulated within the arterial wall. Arterial cells were isolated from carotid arteries of apoE−/− mice at 3 days after wire injury using an enzymatic disaggregation method as previously reported.33Hay C Micko C Prescott MF Liau G Robinson K De Leon H Differential cell cycle progression patterns of infiltrating leukocytes and resident cells after balloon injury of the rat carotid artery.Arterioscler Thromb Vasc Biol. 2001; 21: 1948-1954Crossref PubMed Scopus (18) Google Scholar, 34Galkina E Kadl A Sanders J Varughese D Sarembock IJ Ley K Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent.J Exp Med. 2006; 203: 1273-1282Crossref PubMed Scopus (351) Google Scholar The number of cells obtained from a pool of five common carotid arteries ranged from 3 × 105 to 5 × 105. Cell suspensions were washed twice with phosphate-buffered saline containing 3% bovine serum albumin (BSA) and divided into aliquots (approximately 1 × 105 cells per tube). All fluorescence conjugated antibodies were obtained from BD PharMingen unless specified otherwise. For assessing total leukocyte content, cells were stained for 15 minutes at 4°C with the allophycocyanin-conjugated anti-mouse CD45 mAb (clone 30-F11, 1/450) or an isotype control rat IgG2b,κ. For assessing leukocyte subpopulations, cells were simultaneously stained with the allophycocyanin-conjugated anti-mouse CD45 mAb and either fluorescein isothiocyanate (FITC)-conjugated anti-mouse LY-6G mAb (for granulocytes/neutrophils, clone 1A8, 1/100), or FITC-conjugated anti-mouse Mac-3 (for macrophages, clone M3/84, 1/50, Santa Cruz). To exclude dead cells, 20 μl of propidium iodide (40 μg/ml, Calbiochem) was added to each aliquot immediately before flow cytometry. Propidium iodide-stained cells were excluded from further analysis. Samples were analyzed with a Becton Dickinson FACSCalibur flow cytometer. Data were expressed as percentage of total leukocytes and individual subpopulations in total live cells counted.Blood Collection and Biochemical AnalysisBlood samples were harvested by cardiac puncture into EDTA-containing Microtainer tubes (Becton-Dickinson) at the time of euthanasia. Complete blood counts and automated differential leukocyte counts were performed by the University of Virginia Clinical Pathology Laboratory. For lipoprotein levels, blood samples at the time of death were drawn by cardiac puncture and placed in serum separator tubes. Lipid levels were determined by the University of Virginia Clinical Pathology Laboratory. Fasting blood glucose was measured by glucometer (Accucheck Advantage; Roche Diagnostics, Indianapolis, IN). Plasma sCD40L was measured by a mouse soluble CD40L enzyme-linked immunosorbent assay kit (AXXORA). The lower limit of detection for the sCD40L assay was 0.14 ng/ml, and the overall intra-assay coefficient of variation was 6.5%. The specificity of the sCD40L assay was confirmed by using plasma obtained from CD40L−/− mice in which sCD40L was undetectable by the enzyme-linked immunosorbent assay.In some experiments, the presence of plasma sCD40L was detected by immunoprecipitation and Western blot analysis. Briefly, whole blood (0.8–1.0 ml per mouse) was collected from anesthetized mice by cardiac puncture and anticoagulated with 3.8% trisodium citrate (1:10 vol). Anticoagulated blood was centrifuged immediately at 14,000 rpm for 5 minutes in an Eppendorf microfuge. Supernatant plasma was collected, pooled (>1.0 ml, from three mice), and stored at −80°C until analysis. The pooled plasma was subjected to immunoprecipitation for CD40L with a polyclonal antibody against CD40L (K-19, Santa Cruz) and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, probed with purified MR1 mAb, and visualized with the enhanced chemiluminescence system (Amersham).Preparation of Mouse Neutrophils and PlateletsPeritoneal neutrophils were prepared from mice as previously described.23Simon DI Chen Z Xu H Li CQ Dong J McIntire LV Ballantyne CM Zhang L Furman MI Berndt MC Lopez JA Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18).J Exp Med. 2000; 192: 193-204Crossref PubMed Scopus (499) Google Scholar Mice were injected with 2.5 ml of sterile 3% thioglycolate broth solution (Sigma Chemical Co.) by intraperitoneal administration, and 5 hours after injection neutrophils were harvested and purified using a two-layer Percoll gradient (80% over 64%). Cytospin preparations stained with Giemsa revealed that >90% of the cells were neutrophils. Cell viability (>95%) was assessed using trypan blue exclusion. Isolated neutrophils were resuspended in RPMI 1640 containing 0.5% (w/v) low-endotoxin BSA.Platelet-rich plasma and washed platelets were prepared from mice as previous described.35Elzey BD Tian J Jensen RJ Swanson AK Lees JR Lentz SR Stein CS Nieswandt B Wang Y Davidson BL Ratliff TL Platelet-mediated modulation of adaptive immunity: a communication link between innate and adaptive immune compartments.Immunity. 2003; 19: 9-19Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Washed platelets were resuspended in Tyrode's buffer (10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 12 mmol/L NaHCO3, 137 mmol/L NaCl, 2.7 mmol/L KCl, 0.42 mmol/L NaH2PO4, and 5.5 mmol/L glucose, pH 7.4) containing 0.1% BSA. Platelet count was adjusted to 2 × 108/ml.Detection of CD40 Receptor Expression in Mouse NeutrophilsFor detection of CD40 mRNA expression, total cellular RNA was extracted from fresh isolated mouse peritoneal neutrophils with TRIzol (Invitrogen, Carlsbad, CA) treated with DNase I to remove genomic DNA, then purified with the use of a RNA purification kit (Invitrogen), and reverse-transcribed using the Superscript II (Life Technologies Inc., Rockville, MD) and oligo-dT primers. The following mouse polymerase chain reaction primers36Heinzel FP Rerko RM Hujer AM Underproduction of interleukin-12 in susceptible mice during progressive leishmaniasis is due to decreased CD40 activity.Cell Immunol. 1998; 184: 129-142Crossref Scopus (47) Google Scholar were used: CD40 forward, 5′-TCCCTGCCCAGTCGGCTTCT-3′, and reverse, 5′-CTGTCTTGGCTCATCTCAAA-3′ (505 bp); β-actin forward, 5′-GTGGGCCGCTCTAGGCACCAA-3′, and reverse, 5′-CTCTTTGATGTCACGCACGATTTC-3′ (540 bp). Polymerase chain reaction was performed by an initial cycle at 95°C for 4 minutes followed by 30 cycles for CD40 or 18 cycles for β-actin of 94°C for 45 seconds, 58°C for 45 seconds, 72°C for 1 minute, and ending with a 5-minute extension at 72°C. The amplified products were separated on 1.5% agarose gels.For detection of CD40 protein expression, cellular lysates were prepared from fresh isolated mouse peritoneal neutrophils with RIPA buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 0.5% sodium deoxycholate). Protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL). Equal amounts (20 μg/lane) of protein lysates were run out on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Amersham). Membranes were blocked with blocking solution (Tris-buffered saline/Tween 20 containing 5% nonfat dry milk), blotted with a goat polyclonal CD40 antibody (T-20, Santa Cruz) and visualized with the enhanced chemiluminescence system (Amersham).Flow Cytometry Analysis of Neutrophil Mac-1 Expression and Oxidative BurstIsolated neutrophils (1 × 106/ml, in RPMI 1640/0.1% BSA) were incubated with 3 μl of FITC-conjugated rat anti-mouse CD11b mAb (M1/70 against integrin αM chain of Mac-1), or control FITC-rat IgG2b,κ, for 30 minutes at room temperature in the dark. Neutrophils were then incubated with rm-CD40L in the presence or absence of anti-CD40 mAb (HM40–3), anti-CD40L mAb (MR1), or hamster IgG (Iso) for 15 minutes at 37°C. Neutrophils incubated with phorbol 12-myristate 13-acetate (50 ng/ml) for 5 minutes served as a positive control. Samples were then fixed in 1% buffered paraformaldehyde and analyzed for neutrophil αM integrin expression by a Becton Dickinson FACScalibur with CellQuest software.Neutrophil oxidative burst was measured by a flow cytometric approach using dihydrorhodamine 123 (DHR123) as a fluorescent probe as previously described.37Li G Keenan AC Young J Fierschnaller V Berlin H Hall M Steinhubl SR Smyth SS Effects of unfractionated heparin and glycoprotein IIb/IIIa antagonists versus bivalirdin on myeloperoxidase release from neutrophils.Arterioscler Thromb Vasc Biol. 2007; 27: 1850-1856Crossref PubMed Scopus (27) Google Scholar Briefly, neutrophils were isolated from blood of adult human healthy donors and suspended in Hanks' balanced salt solution containing 0.25% BSA. Aliquots of neutrophils (2 × 105 cells in 200 μl) were loaded with 1 μmol/L DHR123 (Molecular Probes) for 5 minutes at 37°C and then stimulated with rh-CD40L under defined conditions. Samples were analyzed immediately by flow cytometry. Data are expressed as mean fluorescence intensity in FL-1 channel for
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