Activated Neutrophils Induce Epithelial Cell Apoptosis Through Oxidant-Dependent Tyrosine Dephosphorylation of Caspase-8
2014; Elsevier BV; Volume: 184; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2013.12.031
ISSN1525-2191
AutoresSong Jia, Jean Parodo, Emmanuel Charbonney, Jennifer Tsang, Sang Yang Jia, Ori D. Rotstein, András Kapùs, John C. Marshall,
Tópico(s)Immune Response and Inflammation
ResumoActivated neutrophils can injure host cells through direct effects of oxidants on membrane phospholipids, but an ability to induce apoptotic cell death has not previously been reported. We show that neutrophils activated in vivo in patients who have sustained multiple trauma or in vitro by exposure to bacterial lipopolysaccharide promote epithelial cell apoptosis through SHP-1–mediated dephosphorylation of epithelial cell caspase-8. Epithelial cell apoptosis induced by circulating neutrophils from patients who had sustained serious injury depended on the generation of neutrophil-derived reactive oxygen intermediates and was blocked by inhibition of NADPH oxidase or restoration of intracellular glutathione. Caspase-8 was constitutively tyrosine phosphorylated in a panel of resting epithelial cells, but underwent SHP-1–dependent dephosphorylation in response to hydrogen peroxide, activated neutrophils, or inhibition of Src kinases. Cells transfected with a mutant caspase-8 in which tyrosine residues at Tyr397 or Tyr465 are replaced by nonphosphorylatable phenylalanine underwent accelerated apoptosis, whereas either mutation of these residues to phosphomimetic glutamic acid or transfection with the Src kinases Lyn or c-Src inhibited hydrogen peroxide–induced apoptosis. Exposure to either hydrogen peroxide or lipopolysaccharide-stimulated neutrophils increased phosphorylation and activity of the phosphatase SHP-1, increased activity of caspases 8 and 3, and accelerated epithelial cell apoptosis. These observations reveal a novel mechanism for neutrophil-mediated tissue injury through oxidant-dependent, SHP-1–mediated dephosphorylation of caspase-8 resulting in enhanced epithelial cell apoptosis. Activated neutrophils can injure host cells through direct effects of oxidants on membrane phospholipids, but an ability to induce apoptotic cell death has not previously been reported. We show that neutrophils activated in vivo in patients who have sustained multiple trauma or in vitro by exposure to bacterial lipopolysaccharide promote epithelial cell apoptosis through SHP-1–mediated dephosphorylation of epithelial cell caspase-8. Epithelial cell apoptosis induced by circulating neutrophils from patients who had sustained serious injury depended on the generation of neutrophil-derived reactive oxygen intermediates and was blocked by inhibition of NADPH oxidase or restoration of intracellular glutathione. Caspase-8 was constitutively tyrosine phosphorylated in a panel of resting epithelial cells, but underwent SHP-1–dependent dephosphorylation in response to hydrogen peroxide, activated neutrophils, or inhibition of Src kinases. Cells transfected with a mutant caspase-8 in which tyrosine residues at Tyr397 or Tyr465 are replaced by nonphosphorylatable phenylalanine underwent accelerated apoptosis, whereas either mutation of these residues to phosphomimetic glutamic acid or transfection with the Src kinases Lyn or c-Src inhibited hydrogen peroxide–induced apoptosis. Exposure to either hydrogen peroxide or lipopolysaccharide-stimulated neutrophils increased phosphorylation and activity of the phosphatase SHP-1, increased activity of caspases 8 and 3, and accelerated epithelial cell apoptosis. These observations reveal a novel mechanism for neutrophil-mediated tissue injury through oxidant-dependent, SHP-1–mediated dephosphorylation of caspase-8 resulting in enhanced epithelial cell apoptosis. Patients who have sustained severe traumatic injuries develop multiple abnormalities of immunological and metabolic homeostasis, including systemic activation of polymorphonuclear neutrophils (PMNs)1Brown K.A. Brain S.D. Pearson J.D. Edgeworth J.D. Lewis S.M. Treacher D.F. Neutrophils in development of multiple organ failure in sepsis.Lancet. 2006; 368: 157-169Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar and enhanced apoptosis of epithelial cells and lymphocytes.2Hotchkiss R.S. Nicholson D.W. Apoptosis and caspases regulate death and inflammation in sepsis.Nat Rev Immunol. 2006; 6: 813-822Crossref PubMed Scopus (594) Google Scholar The mechanisms that promote apoptosis in acutely ill patients are poorly characterized. Increased circulating levels of soluble Fas ligand3Imai Y. Parodo J. Kajikawa O. de Perrot M. Fischer S. Edwards V. Cutz E. Liu M. Keshavjee S. Martin T.R. Marshall J.C. Ranieri V.M. Slutsky A.S. Injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome.JAMA. 2003; 289: 2104-2112Crossref PubMed Scopus (550) Google Scholar, 4Wesche-Soldato D.E. Chung C.S. 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Pearson J.D. Edgeworth J.D. Lewis S.M. Treacher D.F. Neutrophils in development of multiple organ failure in sepsis.Lancet. 2006; 368: 157-169Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 7Smith J.A. Neutrophils, host defense, and inflammation: a double-edged sword.J Leukoc Biol. 1994; 56: 672-686Crossref PubMed Scopus (750) Google Scholar, 8Weiss S.J. Tissue destruction by neutrophils.N Engl J Med. 1989; 320: 365-376Crossref PubMed Scopus (3812) Google Scholar, 9Nathan C. Ding A. Nonresolving inflammation.Cell. 2010; 140: 871-882Abstract Full Text Full Text PDF PubMed Scopus (1350) Google Scholar Cellular damage has been attributed to direct injury by neutrophil proteases such as elastase,10Smedly L.A. Tonnesen M.G. Sandhaus R.A. Haslett C. Guthrie L.A. Johnston Jr., R.B. Henson P.M. Worthen G.S. Neutrophil-mediated injury to endothelial cells. Enhancement by endotoxin and essential role of neutrophil elastase.J Clin Invest. 1986; 77: 1233-1243Crossref PubMed Scopus (476) Google Scholar to the effects of neutrophil-derived oxidants on lipids in cell membranes, resulting in the necrotic death of the target cell,11Ramaiah S.K. Jaeschke H. Role of neutrophils in the pathogenesis of acute inflammatory liver injury.Toxicol Pathol. 2007; 35: 757-766Crossref PubMed Scopus (249) Google Scholar and to the formation of neutrophil extracellular traps through the extrusion of nuclear chromatin.12Kaplan M.J. Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity.J Immunol. 2012; 189: 2689-2695Crossref PubMed Scopus (677) Google Scholar However, extensive necrotic cell death is uncommon in tissues from patients suffering the sequelae of traumatic injury,13Hotchkiss R.S. Swanson P.E. Freeman B.D. Tinsley K.W. Cobb J.P. Matuschak G.M. Buchman T.G. Karl I.E. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.Crit Care Med. 1999; 27: 1230-1251Crossref PubMed Scopus (1033) Google Scholar and experimental inflammatory lung injury is characteristically accompanied by evidence of concomitant activation of apoptosis.14Mantell L.L. Kazzaz J.A. Xu J. Palaia T.A. Piedboeuf B. Hall S. Rhodes G.C. Niu G. Fein A.F. Horowitz S. Unscheduled apoptosis during acute inflammatory lung injury.Cell Death Differ. 1997; 4: 600-607Crossref PubMed Scopus (49) Google Scholar, 15Kawasaki M. Kuwano K. Hagimoto N. Matsuba T. Kunitake R. Tanaka T. Maeyama T. Hara N. Protection from lethal apoptosis in lipopolysaccharide-induced acute lung injury in mice by a caspase inhibitor.Am J Pathol. 2000; 157: 597-603Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar The capacity of activated neutrophils to induce apoptotic cell death has not previously been demonstrated, although oxidants such as superoxide anion and hydrogen peroxide (products of neutrophil NADPH oxidase) are potent proapoptotic stimuli.16Mayadas T.N. Cullere X. Neutrophil beta2 integrins: moderators of life or death decisions.Trends Immunol. 2005; 26: 388-395Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar The catalytic activity of caspase-8, the apical caspase of the extrinsic pathway of apoptosis, is regulated through the phosphorylation of tyrosine residues at Tyr397 and Tyr465.17Cursi S. Rufini A. Stagni V. Condò I. Matafora V. Bachi A. Bonifazi A.P. Coppola L. Superti-Furga G. Testi R. Barilà D. Src kinase phosphorylates caspase-8 on Tyr380: a novel mechanism of apoptosis suppression.EMBO J. 2006; 25: 1895-1905Crossref PubMed Scopus (162) Google Scholar, 18Jia S.H. Parodo J. Kapus A. Rotstein O.D. Marshall J.C. Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8.J Biol Chem. 2008; 283: 5402-5413Crossref PubMed Scopus (73) Google Scholar Caspase-8 contains an immunoreceptor tyrosine-based inhibition motif-like domain (IxYxxL)19Daëron M. Jaeger S. Du Pasquier L. Vivier E. Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future.Immunol Rev. 2008; 224: 11-43Crossref PubMed Scopus (253) Google Scholar at Tyr310 in the p18 active fragment of the enzyme.20Daigle I. Yousefi S. Colonna M. Green D.R. Simon H.U. Death receptors bind SHP-1 and block cytokine-induced anti-apoptotic signaling in neutrophils.Nat Med. 2002; 8: 61-67Crossref PubMed Scopus (157) Google Scholar Binding of the membrane-associated tyrosine phosphatase SHP-1 to this motif results in SHP-1 mediated dephosphorylation of Tyr397 and Tyr465, followed by activational cleavage of caspase-8, activation of caspase-3, and the progression of apoptosis.18Jia S.H. Parodo J. Kapus A. Rotstein O.D. Marshall J.C. Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8.J Biol Chem. 2008; 283: 5402-5413Crossref PubMed Scopus (73) Google Scholar Because in vitro exposure of A549 lung epithelial cells to hydrogen peroxide can activate p53 and increase caspase-3 activity,21Dandrea T. Hellmold H. Jonsson C. Zhitotovsky B. Hofer T. Wärngård L. Cotgreave I. The transcriptosomal response of human A549 lung cells to a hydrogen peroxide-generating system: relationship to DNA damage, cell cycle arrest, and caspase activation.Free Radic Biol Med. 2004; 36: 881-896Crossref PubMed Scopus (56) Google Scholar we hypothesized that oxidants from activated neutrophils could induce epithelial cell apoptosis through the dephosphorylation of epithelial cell caspase-8. Here, we show that neutrophils harvested from patients who have sustained multiple trauma induce the apoptosis of cultured epithelial cells through the generation of reactive oxygen intermediates. Exposure of epithelial cells to neutrophils from trauma patients, neutrophils activated in vitro by exposure to lipopolysaccharide (LPS), or hydrogen peroxide resulted in increased activity of the phosphatase SHP-1, leading to dephosphorylation of caspase-8 and activation of the extrinsic pathway of apoptosis. These observations reveal a novel mechanism through which neutrophils can induce cellular injury during acute inflammation. We recruited patients with multisystem trauma (Injury Severity Score ISS > 16) who were admitted to the Trauma-Neurosurgical Intensive Care Unit of St. Michael's Hospital. The protocol was approved by the Human Ethics Review Committee of St. Michael's Hospital, and written informed consent was obtained from each patient or a surrogate decision-maker. Circulating neutrophils were obtained from patients or from consenting healthy volunteers by density gradient centrifugation using Ficoll–Hypaque (GE Healthcare, Little Chalfont, UK) as described previously.22Jia S.H. Li Y. Parodo J. Kapus A. Fan L. Rotstein O.D. Marshall J.C. Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis.J Clin Invest. 2004; 113: 1318-1327Crossref PubMed Scopus (531) Google Scholar Cells were resuspended in polypropylene tubes in Dulbecco’s modified Eagle’s medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution. A549 human lung carcinoma cells (CCL-185; ATCC, Manassas, VA) and human embryonic kidney HEK293 cells (CRL-1573; ATCC) were cultured in supplemented DMEM. Human distal airway BEAS-2B cells (CRL-9609; ATCC) were cultured in bronchial epithelial cell basal medium (BEBM; Lonza Canada, Shawinigan, QC, Canada; Lonza, Walkersville, MD) supplemented with bronchial epithelial cell growth medium (BEGM SingleQuot, CC-4175; Lonza Canada) containing bovine pituitary extract, insulin, hydrocortisone, GA-1000 (gentamicin, amphotericin B), epinephrine, retinoic acid, transferrin, and triiodothyronine. Cells were cultured in tissue culture plates (100 × 20 mm), and experiments were performed at 80% confluency. Antibodies used in these studies were murine monoclonal (mAb) anti–caspase-8 (1:1000; Calbiochem; Millipore, Billerica, MA), agonistic anti-CD95 mAb CH11 (100 ng/mL; BioSource International; Life Technologies), murine mAb anti-phosphotyrosine (1:2000; Upstate; Millipore), and (from Santa Cruz Biotechnology, Dallas, TX) anti–caspase-3 (1:1000), murine mAb anti-Lyn (1:1000), anti–C-Src (1:1000), murine mAb anti–SHP-1 (1:1000), murine mAb anti–β-actin (1:4000), and murine mAb anti–c-Myc (1:1000). LPS from Escherichia coli O111:B4 (Sigma-Aldrich, Mississauga, ON, Canada; St. Louis, MO) was used at a concentration of 1 μg/mL. Recombinant TNF-α (BioSource International; Life Technologies) was used at a concentration of 50 ng/mL. The Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (Calbiochem; Millipore) was used at a concentration of 10 μmol/L. Hydrogen peroxide (Sigma-Aldrich) was used at a concentration of 100 μmol/L. Neutrophils were added to epithelial cell cultures at a ratio of 5:1 (2.5 × 106 PMNs to 5 × 105 epithelial cells) and were incubated at 37°C with or without 1 μg/mL LPS for a further 18 hours. Plates were then washed three times with supplemented medium to remove PMNs, and epithelial cell cultures were trypsinized to detach cells from the culture plates. Total RNA from neutrophils from healthy human volunteers was extracted using TRIzol reagent and 1 μg RNA was transcribed to first-strand cDNA using a SuperScript II system (Life Technologies); the resultant cDNA was amplified by PCR using an Expand high-fidelity PCR system (Roche Diagnostics, Indianapolis, IN). Primer sequences are listed in Table 1. Amplified fragments were cloned into the pcDNA3.1/myc-His vector (Life Technologies) according to the manufacturer’s instructions. The recombinant plasmids were transfected into DH5a-competent cells (Life Technologies), and colonies were identified by restriction endonuclease digestion and sequencing.Table 1Primers for Plasmid ConstructionTargetPrimer descriptionSequenceCaspase-8Upstream (contains a HindIII site and a Kozak sequence)5′-GCAAGCTTGCCACCATGGACTTCAGCAGAAATCT-3′Downstream (contains an XbaI site)5′-CTCTAGAATCAGAAGGGAAGACAAGT-3′SHP-1Upstream (contains a HindIII site and a Kozak sequence)5′-GCAAGCTTGCCACCATGGTGAGGTGGTTTCACCGA-3′Downstream (contains an XbaI site)5′-GCTCTAGACTTCCTCTTGAGGGAACCCT-3′LynUpstream (contains a BamHI site and a Kozak sequence)5′-GCGGATCCGCCACCATGGGATGTATAAAATCAAAA-3′Downstream primer (contains an XbaI site)5′-GCTCTAGAAGGCTGCTGCTGGTATTGCCCT-3′Caspase-8 Y→F Tyr397Upstream5′-GGAGCAACCCTTTTTAGAAATGG-3′Downstream5′-CCATTTCTAAAAAGGGTTGCTCC-3′ Tyr465Upstream5′-GAAGTGAACTTTGAAGTAAGC-3′Downstream5′-GCTTACTTCAAAGTTCACTTC-3′Caspase-8 Y→E Tyr397Upstream5′-GGAGCAACCCGAGTTAGAAATGG-3′Downstream5′-CCATTTCTAACTCGGGTTGCTCC-3′ Tyr465Upstream5′-GAAGTGAACGAGGAAGTAAGC-3′Downstream5′-GCTTACTTCCTCGTTCACTTC-3′Caspase-8 Y377SUpstream5′-ATTCAGGCTAGTCAGGGGG-3′Downstream5′-CCCCCTGACTAGCCTGAAT-3′Underline indicates specific amino acid mutation. Open table in a new tab Underline indicates specific amino acid mutation. Tyrosine residues in the caspase-8 molecule were mutated using site-directed primers to mutate the tyrosine residue (TAT or TAC) to phenylalanine (TTT or TTC). Pseudophosphorylation mutants of the same sites were generated by mutating the relevant tyrosine (Y) residues (TAT or TAC) to glutamic acid (E; GAG).23Necula M. Kuret J. Site-specific pseudophosphorylation modulates the rate of tau filament dissociation.FEBS Lett. 2005; 579: 1453-1457Crossref PubMed Scopus (34) Google Scholar Finally, a catalytically inactive mutant of caspase-8 was generated by mutating cysteine (C) 377 to serine (S).24Hasegawa Imamura R. Kinoshita T. Matsumoto N. Masumoto J. Inohara N. Suda T. ASC-mediated NF-kappaB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP.J Biol Chem. 2005; 280: 15122-15130Crossref PubMed Scopus (53) Google Scholar Primer sequences are listed in Table 1. Mutations were performed using a QuikChange site-directed mutagenesis kit (Stratagene; Agilent Technologies, Santa Clara, CA) and a caspase-8/myc-his plasmid as a template to perform the mutant strand synthesis reaction. After DpnI digestion of the amplified product, the mutant DNA was transfected to XL1-Blue supercompetent cells, and colonies were identified by restriction endonuclease digestion and sequencing. Cells were transfected with plasmids containing wild-type or mutant caspase-8 as described previously.18Jia S.H. Parodo J. Kapus A. Rotstein O.D. Marshall J.C. Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8.J Biol Chem. 2008; 283: 5402-5413Crossref PubMed Scopus (73) Google Scholar In brief, 4 μg plasmid and 10 μL FuGENE 6 reagent (Roche Diagnostics) in 200 μL of serum-free medium were added to A549 cell cultures in 10-cm plates at 60% confluency. Cells were cultured for 24 hours and then were washed and cultured for a further 48 hours before study. Epithelial cells were detached from culture plates by trypsinization, washed with PBS, and resuspended in supplemented medium. Cells were permeabilized by the addition of Triton X-100 and incubated for 10 minutes in the dark with 50 μg/mL propidium iodide. The characteristic DNA fragmentation of apoptosis was quantified as the percentage of hypodiploid DNA in permeabilized cells by flow cytometry using a BD FACSCanto cytofluorometer with BD FACSDiva software version 5.0.1 (BD Biosciences, San Jose, CA).25Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunol Methods. 1991; 139: 271-276Crossref PubMed Scopus (4401) Google Scholar A minimum of 10,000 events were recorded. Alternatively, early events during the expression of apoptosis were assessed by detecting exteriorized phosphatidyl serine by flow cytometry as the binding of annexin V, as described previously.22Jia S.H. Li Y. Parodo J. Kapus A. Fan L. Rotstein O.D. Marshall J.C. Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis.J Clin Invest. 2004; 113: 1318-1327Crossref PubMed Scopus (531) Google Scholar Cells were lysed in lysis buffer (10 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; 5 mmol/L EDTA; 1% Triton X-100; 10 mmol/L NaF; 1 mmol/L phenylmethylsulfonyl fluoride; 1 mmol/L Na3VO4; 10 μg/mL leupeptin; 10 μg/mL aprotinin). Lysates were resolved on a 10% SDS-PAGE gel, transferred to nitrocellulose (Amersham; GE Healthcare), and probed with the appropriate primary antibody. Bands were detected with a horseradish peroxidase–conjugated second antibody at a dilution of 1:4000 using an ECL Western blotting detection system (Amersham; GE Healthcare). Blots were stripped and reprobed with an anti-actin antibody to confirm equal loading. For immunoprecipitation studies, cell lysates were centrifuged at 12,000 × g for 10 minutes. Supernatants were precleared with protein G Sepharose beads (Amersham; GE Healthcare) for 1 hour, then were centrifuged again to remove the beads. Protein concentration in the lysates was measured using a bicinchoninic acid protein assay (Pierce BCA; Thermo Fisher Scientific, Waltham, MA). Next, 5 μL of anti–caspase-8 (Calbiochem; Millipore), or 15 μL of anti–SHP-1 (Santa Cruz Biotechnology), or 5 μL of anti-Lyn (Santa Cruz Biotechnology) was added to 500 μL of supernatant and incubated for 1 hour at 4°C before addition of 20 μL protein G beads and incubation for an additional 1 hour or overnight. Suspensions were centrifuged and beads were washed three times in PBS, then boiled in Laemmli buffer for 5 minutes before SDS-PAGE and Western blot analysis. Western blot analysis of immunoblots used anti-phosphotyrosine (clone 4G10) (1:1000; Upstate; Millipore), or other antibodies as noted. Glutathione (GSH) levels were assayed using a 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)–based assay as described previously.26Watson R.W.G. Rotstein O.D. Jimenez M. Parodo J. Marshall J.C. Augmented intracellular glutathione inhibits Fas-triggered apoptosis of activated human neutrophils.Blood. 1997; 89: 4175-4181PubMed Google Scholar A549 cells (5 × 106/mL) were sonicated for 30 seconds on ice in 300 μL of 5% 5-sulfosalicylic acid. After centrifugation for 10 minutes at 10,000 × g, nonprotein sulfhydryls in the supernatant were quantified as the reduction of DTNB by its conversion to 5-thiol-2 nitrobenzoic acid (TNB), measured at 412 nm using a spectrophotometer. GSH levels were expressed as nmol/100 μL per microgram protein by comparison with a standard curve. Caspase activity was determined using a caspase assay kit (BioSource International; Life Technologies). Cell lysates were incubated overnight with 25 μL of synthetic substrate that is preferentially cleaved by caspase-3 (Ac-DEVD-pNA) or caspase-8 (Ac-IETD-pNA). Release of the colorimetric substrate was measured at 405 nm in 96-well plates using a colorimetric plate reader (Titertek-Berthold, Huntsville, AL), and expressed as absorbance at 405 nm per milligram protein. The release of inorganic phosphate from phosphopeptides was measured using a malachite green assay (Upstate; Millipore). In brief, 10 × 106 A549 cells were lysed for 10 minutes on ice in lysis buffer containing 1 mmol/L Na3VO4 and protease inhibitors. Suspensions were centrifuged for 15 minutes at 12,000 × g, protein content of the resulting supernatant was determined, and 500 μL supernatant was incubated with 10 μL anti–SHP-1 antibody (Santa Cruz Biotechnology) prebound to Protein A-Agarose (Santa Cruz Biotechnology) for 3 hours at 4°C. Immunoprecipitates were washed six times in wash buffer (10 mmol/L Tris, pH 7.4), then were incubated with tyrosine phosphopeptide substrate (RRLIEDAEpYAARG) in 10 mmol/L Tris, pH 7.4, for 30 minutes. The reaction was stopped with 100 μL malachite green solution. Absorbance was measured at 620 nm using a LabSystems Multiskan plate reader (LabSystems, Vantaa, Finland; Thermo Fisher Scientific) and phosphate release in pmol phosphate per minute per microgram was determined by comparing absorbance to a standard curve. A549 cells were grown on coverslips in 75-mm plates, and transfected with caspase-8/pcDNA3.1/myc plasmids. After 24 hours, cells were washed with PBS, then fixed on coverslips for 30 minutes with 4% paraformaldehyde. Coverslips were then incubated with 100 mmol/L glycine in PBS, and washed four times; cells were permeabilized with 0.1% Triton X-100 in PBS supplemented with 1% albumin. After blocking with 5% albumin in PBS, coverslips were incubated with primary antibodies (rabbit anti–active caspase-3, 1:1000 and mouse anti–c-Myc, 1:1000) for 60 minutes, washed seven times, and incubated for another 60 minutes with secondary antibody (anti-mouse, Cy3 conjugated, and anti-rabbit, fluorescein isothiocyanate conjugated) then mounted on slides. Slides were washed seven times, and immunofluorescence was detected using an Olympus IX81 microscope (Olympus, Melville, NY; Tokyo, Japan) coupled to an Evolution QEi monochrome camera (Media Cybernetics) using QEDInVivo imaging software version 3.0 and Image-Pro Plus software version 5.1 (Media Cybernetics, Rockville, MD). Intergroup comparisons were performed using one-way analysis of variance followed by Dunnett's t-test, with the α level for statistical significance set at P < 0.05. Studies were repeated a minimum of three times. Data are expressed as means ± SD. We recruited eight trauma patients at a mean of 3 days after injury; demographic characteristics are summarized in Table 2. Circulating neutrophils from these patients showed evidence of activation, reflected in delayed spontaneous apoptosis after 21 hours of in vitro culture (trauma patients, 23.5 ± 5.9% apoptosis; healthy control subjects, 54.2 ± 7.5% apoptosis; P < 0.001). Coculture of patient neutrophils with subconfluent cultures of A549 or HEK293 epithelial cells resulted in increased epithelial cell apoptosis, measured as increased hypodiploid DNA in permeabilized cells (Figure 1A); rates of epithelial cell apoptosis correlated inversely with the magnitude of delay in constitutive neutrophil apoptosis (Figure 1B). The addition of resting healthy neutrophils to epithelial cell cultures triggered a modest increase in apoptosis, reflected in both exteriorization of membrane phosphatidylserine (Figure 1C) and nuclear uptake of propidium iodide (Figure 1D). Addition of 1 μg/mL LPS to neutrophil–epithelial cell cocultures resulted in significantly increased rates of epithelial cell apoptosis (Figure 1, C and D). The increased rates of epithelial cell apoptosis resulting from the addition of LPS, but not those associated with the addition of resting PMNs, could be blocked by the addition of 10 μmol/L diphenyleneiodium (DPI), an inhibitor of NADPH oxidase, or by 10 μg/mL catalase, an enzyme that catalyzes the conversion of hydrogen peroxide to water and oxygen (Figure 1E), suggesting a mechanistic role for oxidants derived from activated neutrophils.Table 2Demographic Characteristics of the Study PopulationCharacteristicValueBaseline Age, years ± SD37 ± 15 Males, no. (%)8 (100) Injury severity score ± SD32 ± 8 Units packed red blood cells in first 24 hours, median no. (range)3 (0–31) Time to sampling, hours ± SD73 ± 45Outcomes Time on mechanical ventilation, days ± SD11 ± 7 Time in ICU, days ± SD13 ± 6 Survival (%)8 (100) Open table in a new tab To test the hypothesis that oxidants alone could dephosphorylate caspase-8 and induce epithelial cell apoptosis, we treated cultured epithelial cells with 100 μmol/L H2O2. The A549 cell line was derived from a human lung carcinoma.27Lieber M. Smith B. Szakal A. Nelson-Rees W. Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.Int J Cancer. 1976; 17: 62-70Crossref PubMed Scopus (1003) Google Scholar To exclude the possibility that any effect was a consequence of malignant transformation, we also used a primary human distal airway cell line (BEAS-2B) and a human embryonic kidney cell line (HEK293). Hydrogen peroxide induced a time-dependent increase in apoptosis (Figure 2A) and a reduction in levels of the endogenous antioxidant GSH (Figure 2B). Enhanced apoptosis was accompanied by reduced tyrosine phosphorylation of caspase-8 (Figure 2C) and increased cleavage of procaspases 8 and 3 to their active forms (Figure 2D), confirming that physiological levels of a product of neutrophil NADPH oxidase can replicate the effects of activated neutrophils. We used pharmacological approaches to further probe the role of neutrophil-derived oxidants in inducing epithelial cell apoptosis. Rates of epithelial cell apoptosis were reduced by the addition of 10 mmol/L GSH, an endogenous antioxidant, to cocultures of BEAS-2B epithelial cells and LPS-activated neutrophils (Figure 3A), as well as by the addition of 10 μmol/L DPI, an NADPH oxidase inhibitor, or 10 μg/mL catalase to enhance the degradation of hydrogen peroxide (Figure 1E); similar results were seen with HEK293 cells (data not shown). Treatment of trauma-patient neutrophils with DPI, catalase, or GSH increased epithelial cell caspase-8 tyrosine phosphorylation (Figure 3B), decreased caspase-8 cleavage (Figure 3C), and attenuated the proapoptotic effects of trauma neutrophils (Figure 3D). Caspase-8a can be phosphorylated on Tyr397 (corresponding to Tyr380 in caspase-8b) and on Tyr465; phosphorylation renders caspase-8 resistant to autocatalytic cleavage, inhibiting the progression of apoptosis that has been initiated through the extrinsic pathway.17Cursi S. Rufini A. Stagni V. Condò I. Matafora V. Bachi A. Bonifazi A.P. Coppola L. Superti-Furga G. Testi R. Barilà D. Src kinase phosphorylates caspase-8 on Tyr380: a novel mechanism of apoptosis suppression.EMBO J. 2006; 25: 1895-1905Crossref PubMed Scopus (162) Google Scholar, 18Jia S.H. Parodo J. Kapus A. Rotstein O.D. Marshall J.C. Dynamic regulation of neutrophil survival through tyrosine phosphorylation or dephosphorylation of caspase-8.J Biol Chem. 2008; 283: 5402-5413Crossref PubMed Scopus (73) Google Scholar Caspase-8 is tyrosine phosphorylated in resting A549 cells (Figure 4A). The addition of 1 μg/mL LPS to cultures had no eff
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