Lipoxin A4 Regulates Bronchial Epithelial Cell Responses to Acid Injury
2006; Elsevier BV; Volume: 168; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.051056
ISSN1525-2191
AutoresCaroline Bonnans, Koichi Fukunaga, Marilyn Levy, Bruce D. Levy,
Tópico(s)Chronic Obstructive Pulmonary Disease (COPD) Research
ResumoAspiration of gastric acid commonly injures airway epithelium and, if severe, can lead to respiratory failure from acute respiratory distress syndrome. Recently, we identified cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) as pivotal mediators in vivo for resolution of acid-initiated acute lung injury. To examine protective mechanisms for these mediators in the airway, we developed an in vitro model of acid injury by transiently exposing well-differentiated normal human bronchial epithelial cells to hydrochloric acid. Transmission electron microscopy revealed selective injury to superficial epithelial cells with disruption of cell attachments and cell shedding. The morphological features of injury were substantially resolved within 6 hours. Acid triggered and early marked increases in COX-2 expression and PGE2 production, and acid-induced PGE2 significantly increased epithelial LXA4 receptor (ALX) expression. LXA4 is generated in vivo during acute lung injury, and we observed that nanomolar quantities increased basal epithelial cell proliferation and potently blocked acid-triggered interleukin-6 release and neutrophil transmigration across well-differentiated normal human bronchial epithelial cells. Expression of recombinant human ALX in A549 airway epithelial cells uncovered ALX-dependent inhibition of cytokine release by LXA4. Together, these findings indicate that injured bronchial epithelial cells up-regulate ALX in a COX-2-dependent manner to promote LXA4-mediated resolution of airway inflammation. Aspiration of gastric acid commonly injures airway epithelium and, if severe, can lead to respiratory failure from acute respiratory distress syndrome. Recently, we identified cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) as pivotal mediators in vivo for resolution of acid-initiated acute lung injury. To examine protective mechanisms for these mediators in the airway, we developed an in vitro model of acid injury by transiently exposing well-differentiated normal human bronchial epithelial cells to hydrochloric acid. Transmission electron microscopy revealed selective injury to superficial epithelial cells with disruption of cell attachments and cell shedding. The morphological features of injury were substantially resolved within 6 hours. Acid triggered and early marked increases in COX-2 expression and PGE2 production, and acid-induced PGE2 significantly increased epithelial LXA4 receptor (ALX) expression. LXA4 is generated in vivo during acute lung injury, and we observed that nanomolar quantities increased basal epithelial cell proliferation and potently blocked acid-triggered interleukin-6 release and neutrophil transmigration across well-differentiated normal human bronchial epithelial cells. Expression of recombinant human ALX in A549 airway epithelial cells uncovered ALX-dependent inhibition of cytokine release by LXA4. Together, these findings indicate that injured bronchial epithelial cells up-regulate ALX in a COX-2-dependent manner to promote LXA4-mediated resolution of airway inflammation. The airway mucosa is lined by a continuous epithelium that forms a vital protective barrier. More than a mechanical interface, epithelial cells also have pivotal regulatory roles in inflammation1Nathan C Points of control in inflammation.Nature. 2002; 420: 846-852Crossref PubMed Scopus (2109) Google Scholar and host defense.2Canny G Levy O Furuta GT Narravula-Alipati S Sisson RB Serhan CN Colgan SP Lipid mediator-induced expression of bactericidal/permeability-increasing protein (BPI) in human mucosal epithelia.Proc Natl Acad Sci USA. 2002; 99: 3902-3907Crossref PubMed Scopus (253) Google Scholar Aspiration of gastric acid can directly injure the upper respiratory tract epithelium, leading to disruption of the epithelial barrier, cell shedding, and acute inflammation.3Wynne JW Ramphal R Hood CI Tracheal mucosal damage after aspiration. A scanning electron microscope study.Am Rev Respir Dis. 1981; 124: 728-732PubMed Google Scholar Acid aspiration is a common cause of clinical illness, including acute lung injury and acute respiratory distress syndrome4Ware LB Matthay MA The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4611) Google Scholar—diseases with excess morbidity and mortality and no available treatment. Therefore, identification of bronchial epithelium responses to acid that promote restitution and limit acute inflammation is needed for new therapeutic insights. Acute inflammation is regulated in part by lipid mediators generated from arachidonic acid (AA).1Nathan C Points of control in inflammation.Nature. 2002; 420: 846-852Crossref PubMed Scopus (2109) Google Scholar Proinflammatory eicosanoids—prostaglandins (PGs) and leukotrienes (LTs)—are generated by cyclooxygenases (COXs) and 5-lipoxygenase (5-LO)5Samuelsson B Dahlen SE Lindgren JA Rouzer CA Serhan CN Leukotrienes and lipoxins: structures, biosynthesis, and biological effects.Science. 1987; 237: 1171-1176Crossref PubMed Scopus (2076) Google Scholar to participate in the pathophysiology of acid-induced acute lung injury.6Nagase T Uozumi N Ishii S Kume K Izumi T Ouchi Y Shimizu T Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2.Nat Immunol. 2000; 1: 42-46Crossref PubMed Scopus (265) Google Scholar Resolution of inflammation in acute lung injury is characterized by clearance of neutrophils [polymorphonuclear leukocytes (PMNs)] from the lung and restoration of epithelial barrier function.7Matthay MA Zimmerman GA Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management.Am J Respir Cell Mol Biol. 2005; 33: 319-327Crossref PubMed Scopus (515) Google Scholar Natural resolution of inflammation occurs via the synthesis of braking signals such as lipoxins (LXs) at sites of inflamed tissue.8Gilroy DW Lawrence T Perretti M Rossi AG Inflammatory resolution: new opportunities for drug discovery.Nat Rev Drug Discov. 2004; 3: 401-416Crossref PubMed Scopus (654) Google Scholar LXs are locally produced via cell-cell interactions between leukocytes and resident cells during multicellular host responses to injury, inflammation, and microbial invasion.9Serhan CN Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution.Prostaglandins Leukot Essent Fatty Acids. 2005; 73: 141-162Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar LXs display diverse counterregulatory actions, including inhibition of PMN functional responses, T-cell activation, and cytokine signaling and release, plus stimulation of macrophage clearance of apoptotic PMNs. Together, these properties of LXs serve to promote resolution of acute inflammation.10McMahon B Godson C Lipoxins: endogenous regulators of inflammation.Am J Physiol. 2004; 286: F189-F201Google Scholar LXA4 exerts many of its bioactions through its cognate receptor ALX, which is expressed on leukocytes11Takano T Fiore S Maddox JF Brady HR Petasis NA Serhan CN Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors.J Exp Med. 1997; 185: 1693-1704Crossref PubMed Scopus (393) Google Scholar and airway epithelial cells.12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar Unlike many other tissues, the lung constitutively expresses COX-2,12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar and COX-2-derived mediators promote in vivo resolution in several models of thoracic inflammation, including allergic pleuritis,13Bandeira-Melo C Serra MF Diaz BL Cordeiro RS Silva PM Lenzi HL Bakhle YS Serhan CN Martins MA Cyclooxygenase-2-derived prostaglandin E2 and lipoxin A4 accelerate resolution of allergic edema in Angiostrongylus costaricensis-infected rats: relationship with concurrent eosinophilia.J Immunol. 2000; 164: 1029-1036PubMed Google Scholar carrageenan-induced pleurisy,14Gilroy DW Colville-Nash PR Willis D Chivers J Paul-Clark MJ Willoughby DA Inducible cyclooxygenase may have anti-inflammatory properties.Nat Med. 1999; 5: 698-701Crossref PubMed Scopus (1135) Google Scholar and acid-triggered acute lung injury.12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar COX-2-derived PGE2 triggers eicosanoid class switching in PMN by decreasing 5-LO-catalyzed LT formation and increasing 15-LO expression and LX biosynthesis.15Levy BD Clish CB Schmidt B Gronert K Serhan CN Lipid mediator class switching during acute inflammation: signals in resolution.Nat Immunol. 2001; 2: 612-619Crossref PubMed Scopus (1155) Google Scholar In a murine model of acid-initiated acute lung injury, levels of PGE2, LXA4, and ALX expression increase to dampen lung inflammation and injury, suggesting direct roles for LX signaling in the resolution of airway epithelial injury. Here, we present evidence for regulation of injured human bronchial epithelial cell function by COX-2-dependent increases in epithelial ALX that limit proinflammatory responses to acid and promote a return to homeostasis. LXA4 was obtained from Calbiochem (San Diego, CA). PGE2, the COX-2 selective inhibitor NS398 and anti-COX-2 polyclonal antibody were acquired from Cayman Chemical (Ann Arbor, MI), and human recombinant tumor necrosis factor (TNF)-α, rabbit IgG, and fluorescein isothiocyanate-conjugated anti-rabbit IgG antibodies were from BD Pharmingen (San Diego, CA). Anti-ALX (also named FPRL-1) polyclonal antibody was from Origene (Rockville, MD). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The polyclonal antibody against β-actin was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary normal human bronchial epithelial (NHBE) cells (Clonetics-BioWhittaker, San Diego, CA) were cultured in an air/liquid interface system.12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar In brief, cells were expanded on tissue culture-treated plastic in bronchial epithelial growth medium (Clonetics-BioWhittaker) supplemented with bovine serum albumin (1.5 μg/ml) and retinoic acid (50 nmol/L) and plated on uncoated nucleopore membranes (24-mm diameter, 0.4-μm pore size, Transwell Clear; Costar, Cambridge, MA) in a 1:1 mixture of bronchial epithelial growth medium and Dulbecco's modified Eagle's medium (Invitrogen Corp., Carlsbad, CA) applied at the basal side only to establish an air/liquid interface. Cells were maintained in culture for 21 days to obtain a differentiated cell population with a mucociliary phenotype. In some experiments, NHBE cells were exposed (37°C, 5% CO2) to LXA4 (100 nmol/L, 15 minutes), PGE2 (0.1 or 10 nmol/L, 15 minutes), or NS398 (10 μmol/L, 1 hour) before acid injury. After pH neutralization, fresh medium was added to the bottom chamber. Human type II alveolar epithelial A549 cells were grown in RPMI 1640 medium (Invitrogen Corp.) with 10% heat-inactivated fetal bovine serum (Sigma Aldrich Corp., St. Louis, MO), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Invitrogen Corp.). Full-length recombinant human ALX (rhALX) cDNA was cloned into HindIII/XbaI sites of pcDNA3 vector. A549 cells (6 × 105 cells in 60-mm dishes) were transfected with 5 mg of either pcDNA3 or pcDNA3-hALX using Superfect transfection reagent according to the manufacturer's instructions (Qiagen, Chatsworth, CA). Neomycin-resistant clones were selected (750 mg/ml) and expanded. In some experiments transfected A549 cells were exposed (15 minutes, 37°C, 5% CO2) to LXA4 (0.01 to 10 nmol/L) or vehicle before HCl (0.1 N, pH 2.0) or TNF-α (1 ng/ml). After 6 hours, supernatants were removed and assayed for cytokine release. Hydrochloric acid (HCl, 0.1 N, pH 1.5, 500 μl/well) was gently applied drop-by-drop onto the apical side of NHBE cell cultures and incubated for 5 minutes at 37°C. Then, acid was removed and pH neutralized with phosphate-buffered saline (PBS) (until pH ≥ 7). Fresh medium was added to the bottom of the wells and NHBE cells were returned to 37°C, 5% CO2. The pH of the acid applied to nondifferentiated epithelial cells (ie, A549 cells) was 2.0 rather than 1.5 because these cells were more sensitive to acid injury than the well-differentiated NHBE cells. NHBE cells on inserts were fixed with 2.5% glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in 0.1 mol/L sodium cacodylate buffer for 1 hour at room temperature and kept at 4°C until processing. Cells were then rinsed in cacodylate buffer and postfixed in 1.25% osmium tetroxide (Electron Microscopy Sciences) for 1 hour at room temperature. After rinsing in 15% ethanol, cells were stained in 4% aqueous uranyl acetate (Electron Microscopy Sciences) for 1 hour, dehydrated through graded ethanols, and embedded in Polybed 812 (PolySciences, Inc., Warrington, PA). Tissue was thin sectioned on a Reichert-Jung Ultra Cut (Leica, Inc., Deerfield, IL), poststained in uranyl acetate and lead citrate, viewed on a Zeiss 902 electron microscope (Carl Zeiss SMT, Thornwood, NY), and recorded with Kodak E.M. film (Eastman Kodak Co., Rochester, NY). Mediators were determined in cell supernatants by sensitive and specific enzyme-linked immunosorbent assays for PGE2 (Cayman Chemical), interleukin (IL)-6, and IL-8 (Diaclone, Stamford, CT). Total RNA was purified from cell lysates (RNeasy; Qiagen, Valencia, CA), and cDNA were synthesized (Ready-to-Go RT-PCR beads; Amersham, Piscataway, NJ). Semiquantitative human ALX gene expression was determined using specific primers for human ALX (sense primer, 5′-TT GCT CTA GTC CTT ACC TTG C-3′, and anti-sense primer, 5′-GC AAG TAC AAA ATC ATT GAC ATC-3′) and β-actin (internal control, sense primer, 5′-GCTGGAGCATGCCCGTATT-3′, and anti-sense primer, 5′-ACC CTG CTG TGC TGA GTGTC-3′). After electrophoresis, densitometry was performed using Scion Image software. Quantitative polymerase chain reaction reactions were performed using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in an ABI Prism 5700 (Applied Biosystems). Fold changes were calculated relative to control treatment using the deltadelta Ct method.16Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (133822) Google Scholar β-Actin expression was used as reference standard. The respective forward and reverse primers for COX-2 were 5′-CCT TGA CCA TGA TGG CCA G-3′ and 5′-TGG AGG GCA GTG CTG TTT G-3′. For protein determination, whole cells were lysed in 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, 0.25% deoxycholic acid sodium salt, 1 mmol/L phenyl-methyl sulfonyl fluoride, and protease inhibitors (Complete cocktail tablets; Roche Applied Science, Indianapolis, IN). Total protein (50 μg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% Tris-HCl gels and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were blocked [4% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), 1 hour, room temperature] and probed with rabbit polyclonal anti-human COX-2 antibody (1:1000 dilution) overnight at 4°C. After serial washes with TBST, membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:20,000 dilution). The polyclonal antibody against β-actin (1:500 dilution) was used as internal control. Visualization was performed by chemiluminescence (Pierce Biotechnology, Inc., Rockford, IL) followed by autoradiography. Cell proliferation was determined using reduction of tetrazolium dye (MTT; Chemicon Int., Temecula, CA) to blue formazan to measure living cells. After 24 hours in epidermal growth factor (EGF)-free medium, NHBE cells (∼3 × 103 cells/well) were seeded on 96-well plates and cultured in bronchial epithelial growth medium for 24 hours (37°C, 5% CO2) in the presence of LXA4 (10, 100, and 1000 nmol/L), PGE2 (200 nmol/L), EGF (0.5 ng/ml), or vehicle (medium without EGF). MTT solution (10 μl) was then added to each well and incubated for 4 hours (37°C). Dye was extracted from cells in 100 μl of isopropanol: 1 N HCl (96:4, v:v), and absorbance at 570 nm was determined. A linear relationship was determined for MTT reduction (absorbance at 570 nm) and NHBE cell number (y = 4 × 10−5(x) + 0.0532, r2 = 0.97). NHBE cells were grown on inverted polycarbonate filters with a surface area of 0.33 cm2 (Costar inserts; Costar Corp.) to study basal to apical transmigration.17Parkos CA Delp C Arnaout MA Madara JL Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction.J Clin Invest. 1991; 88: 1605-1612Crossref PubMed Scopus (302) Google Scholar The cells were cultured for 14 days at an air/liquid interface. Human PMNs were isolated from healthy patients who denied taking any medications for at least 2 weeks and had given written informed consent to a protocol approved by Brigham and Women's Hospital's Human Research Committee. PMNs were isolated from whole blood as previously described15Levy BD Clish CB Schmidt B Gronert K Serhan CN Lipid mediator class switching during acute inflammation: signals in resolution.Nat Immunol. 2001; 2: 612-619Crossref PubMed Scopus (1155) Google Scholar and resuspended in modified HBSS (lacking Ca2+ and Mg2+) at 25 × 106 cells/ml. PMNs (1 × 106/well) were exposed to LXA4 (0.1, 1, 10, 100, or 1000 nmol/L) or vehicle for 15 minutes at 37°C before addition to the upper chambers. The chemoattractant LTB4 (1 μmol/L in HBSS containing Ca2+ and Mg2+) was added to the lower chambers, and transmigration was allowed to proceed for 60 minutes (37°C, 5%CO2). PMN number was determined by measurement of MPO activity.17Parkos CA Delp C Arnaout MA Madara JL Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction.J Clin Invest. 1991; 88: 1605-1612Crossref PubMed Scopus (302) Google Scholar A549 cells (500,000 cells) were incubated (4°C, 30 minutes) in fluorescence-activated cell sorting buffer (DPBS + 5% fetal calf serum + 0.01% sodium azide) with 1 mg of rabbit anti-human ALX or with class-matched irrelevant rabbit IgG1. Each sample was washed, resuspended in fluorescence-activated cell sorting buffer containing a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (1:100 final dilution), and incubated for 30 minutes (4°C). After washing, samples were analyzed by cytofluorometer (FACScan; Becton Dickinson, Mountain View, CA) with Cellquest software. Data were collected on 10,000 cells per sample. Values represent the mean ± SEM. Comparisons among groups were performed by Student's t-test. For all analyses, findings were considered significant when P < 0.05. To determine the effect of acid injury on epithelial cells, we examined NHBE cell morphology by transmission electron microscopy. Before acid injury, the NHBE cells displayed typical features of a well-differentiated bronchial epithelium with basal and ciliated cells (Figure 1A) as well as goblet cells that secreted an apical coating of mucus (Figure 1B). Within 5 minutes, acid damaged select superficial cells with nuclear and cytoplasmic changes of necrosis (Figure 1C). Dying cells were released from the apical surface of the epithelium (Figure 1, D and E). Two hours after acid injury, the entire superficial layer of ciliated and goblet cells was apically shed (Figure 1F) with disruption of cellular attachments (Figure 1G). The acid injury did not appear to alter basal cell layer morphology. Within 6 hours the superficial epithelial layer was primarily restored (Figure 1H), albeit goblet cell numbers were still decreased compared to cells cultured in the absence of acid and regeneration of cilia was incomplete (Figure 1I). Because COX-2 is pivotal to resolution in vivo of acid-initiated acute lung injury,12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar we investigated the time-dependent expression and activity of COX-2 in well-differentiated NHBE cells after in vitro acid injury. COX-2 mRNA and protein were significantly increased, with maximal levels occurring 2 hours after acid injury (6.1 ± 1.6-fold increase) and baseline levels returning by 72 hours. COX-2 protein expression was also increased 2 hours after acid injury (Figure 2A, inset). In addition, PGE2 generation was significantly increased within 2 hours (0.2 ± 0.025 ng/ml) and continued to increase during the first 24 hours (0.73 ± 0.17 ng/ml at 24 hours) (Figure 2B). Epithelial PGE2 was COX-2-derived as a COX-2 selective inhibitor completely blocked acid-induced PGE2 release (Figure 2C). Because a component of the in vivo protective effects of COX-2 during acid-mediated acute lung injury occurred via enhanced LX signaling,12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar we next determined NHBE cell ALX expression in vitro after acid injury. ALX mRNA expression significantly increased 2 hours after acid injury (3.7 ± 0.5-fold) and, similar to COX-2, returned to baseline within 72 hours (Figure 3A). To determine whether PGE2 directly modulates ALX expression, NHBE cells were cultured in the presence of PGE2 in amounts similar to those present in NHBE cell supernatants early (within 2 hours) after acid injury (Figure 2B) and those present during in vivo experimental acute lung injury.12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar PGE2 significantly increased ALX mRNA 1.8 ± 0.1- and 4.5 ± 0.7-fold with 0.1 ng/ml and 10 ng/ml PGE2, respectively. To determine whether COX-2-derived PGE2 from NHBE cells can directly increase ALX expression, we exposed NHBE cells to acid in the presence of a selective COX-2 inhibitor (Figure 3C). Acid-induced increases in ALX expression were significantly decreased with the COX-2 inhibitor (69.2 ± 13.4% inhibition). Together, these results indicate that acid triggers NHBE cell expression of ALX in part via COX-2-dependent generation of PGE2. LXA4 is an ALX ligand that is generated in vivo during acute lung injury,12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar so we next examined its impact on NHBE cell functional responses to injury. Because cell proliferation is an early event in restoring airway epithelial integrity, we first assessed the impact of LXA4 on basal NHBE cell proliferation. LXA4 induced a concentration-dependent increase in basal cell number (Figure 4A) that was similar in potency to PGE2 (200 nmol/L) and EGF (0.5 ng/ml), known proliferative agonists for bronchial epithelial cells.18Savla U Appel HJ Sporn PH Waters CM Prostaglandin E(2) regulates wound closure in airway epithelium.Am J Physiol. 2001; 280: L421-L431Google Scholar No significant changes in basal NHBE cell shape or size were observed with LXA4. Because IL-6 is a pleiotropic cytokine produced by epithelial cells during acute inflammation, acute lung injury, and acute respiratory distress syndrome,19Cromwell O Hamid Q Corrigan CJ Barkans J Meng Q Collins PD Kay AB Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumour necrosis factor-alpha.Immunology. 1992; 77: 330-337PubMed Google Scholar, 20Parsons PE Eisner MD Thompson BT Matthay MA Ancukiewicz M Bernard GR Wheeler AP Lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury.Crit Care Med. 2005; 33: 1-6discussion 230–232Crossref PubMed Scopus (602) Google Scholar we next determined the effect of LXA4 on NHBE cell IL-6 release (Figure 4B). Levels of IL-6 were markedly increased in NHBE cell supernatants after acid injury (89.6 ± 11.6 pg/ml compared to untreated cells 6.1 ± 1.9 pg/ml). LXA4 (100 nmol/L) and PGE2 (200 nmol/L) dramatically inhibited IL-6 release from acid-injured NHBE cells (81.5 ± 6.0% inhibition and 59.8 ± 6.1% inhibition, respectively). Incubation of acid-injured NHBE cells with both LXA4 and PGE2 also markedly inhibited IL-6 release (72.3. ± 5.6%), and such inhibition was significantly greater than that seen with PGE2 alone (n ≥ 3, P < 0.05). No significant differences in IL-6 inhibition were observed for the combination of LXA4 and PGE2 compared to LXA4 alone (Figure 4B). LXA4 inhibits PMN transmigration across epithelial monolayers21Colgan SP Serhan CN Parkos CA Delp-Archer C Madara JL Lipoxin A4 modulates transmigration of human neutrophils across intestinal epithelial monolayers.J Clin Invest. 1993; 92: 75-82Crossref PubMed Scopus (217) Google Scholar and PMN accumulation in acid-injured lung in vivo,12Fukunaga K Kohli P Bonnans C Fredenburgh LE Levy BD Cyclooxygenase 2 plays a pivotal role in the resolution of acute lung injury.J Immunol. 2005; 174: 5033-5039PubMed Google Scholar so we next determined its effect on PMN trans-epithelial migration across well-differentiated NHBE cells. Pathophysiological roles have been assigned to the PMN agonist LTB4 in acute lung injury,6Nagase T Uozumi N Ishii S Kume K Izumi T Ouchi Y Shimizu T Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2.Nat Immunol. 2000; 1: 42-46Crossref PubMed Scopus (265) Google Scholar, 22Goldman G Welbourn R Kobzik L Valeri CR Shepro D Hechtman HB Synergism between leukotriene B4 and thromboxane A2 in mediating acid-aspiration injury.Surgery. 1992; 111: 55-61PubMed Google Scholar so we used LTB4 as a PMN chemoattractant for transmigration. LXA4 gave potent and concentration-dependent inhibition of PMN transmigration (Figure 4C). At 0.1 nmol/L, LXA4 displayed 56.9 ± 6.1% inhibition, and maximum inhibition (98.3 ± 2.9%) was achieved with 100 nmol/L LXA4. Together, these results indicate that LXA4 mediates counterregulatory actions on human bronchial epithelial cells by promoting basal cell proliferation and inhibiting proinflam-matory events, such as cytokine release and PMN transmigration. To investigate whether LXA4's anti-inflammatory effects on airway epithelial cells are mediated by its cognate G protein-coupled receptor ALX, we used A549 epithelial cells, which do not express ALX,23Gronert K Gewirtz A Madara JL Serhan CN Identification of a human enterocyte lipoxin A4 receptor that is regulated by interleukin (IL)-13 and interferon gamma and inhibits tumor necrosis factor alpha-induced IL-8 release.J Exp Med. 1998; 187: 1285-1294Crossref PubMed Scopus (194) Google Scholar, 24Bonnans C Mainprice B Chanez P Bousquet J Urbach V Lipoxin A4 stimulates a cytosolic Ca2+ increase in human bronchial epithelium.J Biol Chem. 2003; 278: 10879-10884Crossref PubMed Scopus (59) Google Scholar and generated stable expression of rhALX by transfection (Figure 5). ALX expression was verified in the A549 stable transfectants by reverse transcriptase-polymerase chain reaction (Figure 5A) and flow cytometric analysis, which indicated that rhALX protein was expressed on cell surfaces (Figure 5B). A549 cells with and without ALX expression were exposed to acid in the presence or absence of LXA4. IL-6 release was significantly reduced by LXA4 (0.1 and 1 nmol/L) in cells expressing rhALX, but these concentrations of LXA4 had little effect on acid-injured A549 cells without ALX expression (Figure 5C). In addition to IL-6, TNF-α and IL-8 have also been associated with PMN lung recruitment and activation in acute lung injury,19Cromwell O Hamid Q Corrigan CJ Barkans J Meng Q Collins PD Kay AB Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumour necrosis factor-alpha.Immunology. 1992; 77: 330-337PubMed Google Scholar, 20Parsons PE Eisner MD Thompson BT Matthay MA Ancukiewicz M Bernard GR Wheeler AP Lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury.Crit Care Med. 2005; 33: 1-6discussion 230–232Crossref PubMed Scopus (602) Google Scholar so we determined the impact of ALX expression on TNF-α-induced IL-8 secretion. LXA4 yielded concentration-dependent inhibition of IL-8 release in TNF-α-activated A549 cells expressing rhALX in sharp contrast to cells not expressing the receptor (Figure 5D). LXs are produced locally in the lung to regulate inflammatory cells and promote resolution of acute inflammation. The present results provide the first evidence for potential anti-inflammatory and proresolving roles for LXs on human bronchial epithelial cells. In a new experimental model of acid aspiration injury, well-differentiated NHBE cells engage lipid mediator signaling pathways to regulate cell responses and restore mucosal integrity,
Referência(s)