1Division of Respirology, Department of Medicine, The University of Toronto, Toronto, Ontario, M5S 1A8; 2Department of Pathobiology and Laboratory Medicine and 3Toronto General Hospital Research Institute of the University Health Network, Toronto, Ontario, Canada M5G 2C4
Submitted 13 August 2003 ; accepted in final form 2 March 2004
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ABSTRACT |
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neutrophil; inflammation; caspase; Src; transmigration
The primary function of neutrophils is defense of the host against pathogenic microorganisms during which they release cytotoxic products including proteolytic enzymes and reactive oxygen species that are microbicidal. Paradoxically, these same cellular and biochemical events may also damage host tissues in various organs [e.g., intestine, kidney, and lung (35, 51, 52, 6365)]. In diseases involving inflammation of gastrointestinal epithelium, such as Crohn's disease and ulcerative colitis, neutrophils invade epithelia resulting in epithelial denudation (6) and increased epithelial apoptosis (31).
Although the initiation of cellular apoptosis can be viewed as occurring by two distinct pathways, one involving trimerization of death domain-containing receptors such as Fas (extrinsic pathway) and the other via alterations in mitochondrial membrane permeability (intrinsic pathway), cross-talk can occur between the two pathways (37, 58, 62). The initial caspase in the extrinsic pathway is caspase-8, and in the intrinsic pathway, it is caspase-9, which acts as part of the ternary complex including APAF-1 and cytochrome c. Activation of either of these pathways by cleavage of the initial procaspase results in the downstream activation of caspase-3. Caspase-3 activation, common to both pathways, can then result in activation of caspase-6 or -7 with subsequent activation of caspase-8 (57).
Studies evaluating Fas-mediated apoptosis of intestinal epithelial cells (1) have shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) sensitizes cells to Fas-induced apoptosis, whereas constitutive expression of Akt protects cells from Fas-mediated apoptosis. However, recent in vitro studies (42) in intestinal epithelial cells (T84) demonstrate that chemoattractant-induced polymorphonuclear neutrophil (PMN) transmigration is associated with increased epithelial apoptosis independent of the Fas/Fas ligand pathway. In these latter studies, increased levels of caspases may have resulted from induction of apoptosis following rearrangement of the epithelial actin cytoskeleton associated with neutrophil transmigration.
Akt (PKB) is an important regulator of cell survival and proliferation (30). Two serine-threonine regulatory sites (Thr308 and Ser473) are phosphorylated to activate Akt in a PI3-kinase-dependant manner; PDK1 has been identified as the upstream kinase responsible for phosphorylating Thr308 (30). Recently, two tyrosine phosphorylation (Tyr315 and Tyr326) regulatory sites on Akt have been identified (11). When activated, Akt has different downstream targets including phosphorylation and inactivation of GSK3. In turn, this inhibits GSK3-mediated phosphorylation of -catenin, which normally targets
-catenin for degradation. Free
-catenin can then accumulate in the cytoplasm and translocate to the nucleus resulting in increased transcription of genes important in progression of the cell cycle (4). Akt also inactivates the proapoptotic proteins BAD and caspase-9 by phosphorylation (9, 1315).
In vitro studies evaluating how cell-cell and cell-matrix adhesion regulate cell survival have demonstrated that homotypic interactions between the extracellular adherens junction protein E-cadherin are associated with increased Akt activity (48), whereas decreased Akt activity can occur during apoptotic epithelial cell death secondary to cell detachment from the matrix (anoikis) (3, 33). Studies in mouse embryos deficient in endothelial cadherin (VE-cadherin) (10) have shown an increase in apoptotic endothelial cells, related to decreased phosphorylation of AKT in response to stimulation of vascular endothelial growth factor-A receptor compared with controls.
Exposure to leukocyte elastase can cause apoptosis in endothelial cells (66). Earlier studies by us and others have demonstrated injurious effects of elastase on epithelial barrier function (24, 42). However, the effects of elastase on cell survival are not understood in detail. In the present study, we evaluated the effects of leukocyte elastase on epithelial cell apoptosis and Akt activity. We demonstrated that leukocyte elastase induces apoptosis of epithelial cells via induction of alterations in mitochondrial membrane permeability and via effects on epithelial Akt activity.
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MATERIALS AND METHODS |
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Human T84 cells (ATCC) were grown in a 1:1 mix of DMEM and Ham's F-12 Nutrient Mix (GIBCO, Grand Island, NY) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Cansera International, Rexdale, Ontario) and 2% (vol/vol) penicillin-streptomycin (GIBCO-BRL) and grown at 37°C in 5% CO2. T84 cells were seeded onto inverted polycarbonate filters (1-cm2 surface area, 3.0-µm pore; Costar, Cambridge, MA). After overnight incubation, filters were placed upright in 12-well culture plates so that the epithelial side was on the lower (dependent) side of the filter. Epithelial cells were used when confluent as determined by transepithelial resistance measured with a dual-voltage ohmmeter clamp (World Precision Instruments, Sarasota, FL) (24). When indicated, epithelial cells were incubated with 1) PMN lysates (see Fig. 2), 2) purified leukocyte elastase (Calbiochem-Novobiochem, La Jolla, CA), 3) 5 µM LY294002 (Calbiochem-Novobiochem) to inhibit PI3-kinase, 4) 20 µM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) to inhibit Src tyrosine kinases (Calbiochem-Novobiochem), 5) 5 µM Akt inhibitor to inhibit Akt activity (Calbiochem-Novobiochem), or 6) 100 nM 4',6-diamidino-2-phenyindole (DAPI) (20 min at 37°C; Molecular Probes, Eugene, OR) to identify epithelial cells in the lower chamber of wells.
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Human PMN (>98% pure) were isolated from citrated whole blood by discontinuous plasma-Percoll gradients (29), resuspended in HBSS (GIBCO-BRL) and used immediately after isolation. The integrity and nonactivated state of PMN isolated in this manner have been previously validated (16, 29). In some instances, PMN were incubated with 12 µM Dupont Merck Pharmaceuticals (DMP)-777, a specific inhibitor of PMN elastase (gift from Dr. Robert Vender, Dupont Pharmaceuticals, Wilmington, DE), for 10 min (24). Where indicated, PMN were also incubated with 10 µm 5- (and-6)-{[4-chloromethyl)benzoyl]amino}tetramethylrhodamine (CMTMR) (20 min at 37°C; Molecular Probes) to identify PMN in the lower chamber of wells.
For some experiments, PMN were added to the upper compartment of inserts; transmigration was induced by a chemotactic gradient established with a final concentration of 1 µM N-formylmethionyl-leucyl-phenylalanine (fMLP) in the lower compartment. For other experiments, 10 x 106 PMN were untreated or treated with DMP-777, placed in a microcentrifuge tube, sedimented by centrifugation, and the supernatant discarded. The cells were then subjected to five cycles of freeze/thaw in dry ice and alcohol, following which HBSS was added to the tube and the cells were sonicated with a probe sonicator. After high-speed centrifugation, the supernatant was removed and added to epithelial monolayers as indicated. All inserts were washed and incubated in HBSS for 45 min before the addition of PMN or PMN lysates.
Apoptosis Assays
Epithelial cell apoptosis was measured in several different ways.
Morphometric analysis of epithelial apoptosis. Filters were fixed in 10% formyl alcohol and stained with hematoxylin and eosin. Apoptotic epithelial cells were identified by their standard morphological characteristics on microscopic examination (cytoplasmic condensation and eosinophilia, karyopyknosis, and fragmentation). The measurements were made by an investigator blinded to the treatment protocols of each filter. The number of apoptotic cells is reported as the means ± SE per 10 high-power fields (HPF).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed on cells grown on filters using an in situ death-detection kit (Boerhinger-Mannheim, Mannheim, Germany) according to the manufacturer's instructions. The total number of TUNEL-positive cells was counted in one quarter of each filter (divided before staining) from at least three separate experiments. The mean number of TUNEL-reactive cells was taken as representative of TUNEL staining for each condition. For selected experiments, detached cells (PMN prelabeled with CMTMR and epithelial cells) were collected from the bottom wells, sedimented to a glass slide by centrifugation, and labeled using the TUNEL assay kit. Monolayers were mounted with fluorescent mounting media (DAKO, Carpinteria, CA) onto slides and visualized using a Leica DM-IRB inverted fluorescence microscope. Images were captured with a Princeton Instruments MicroMax cooled charge-coupled device camera under the control of Universal Imaging MetaFluor software.
Mitochondrial membrane potential assay. 1) For experiments involving PMN, epithelial cells were prelabeled with DAPI, and PMN were induced to transmigrate as described above. Epithelial cells that detached 18 h following induction of PMN transmigration were collected from the bottom wells and labeled using the DePsipher kit (Trevigen) according to the manufacturer's instructions. This assay uses a cationic dye that fluoresces red within intact mitochondria (where it assumes a multimeric form) and fluoresces green in the cytoplasm of apoptotic cells. 2) For experiments involving addition of purified elastase, epithelial cells were grown on coverslips and incubated with leukocyte elastase for 3 h before being labeled using the DePsipher kit (Trevigen) according to the manufacturer's instructions.
Flow cytometry. Apoptotic epithelial cells were identified using M30, an antibody against a neoepitope produced during caspase cleavage of cytokeratin 18 that is specific to apoptotic epithelial cells (41, 45). Briefly, to detect epithelial cells committed to apoptosis following exposure to PMN lysates or elastase, adherent cells were removed with trypsin/EDTA and combined with the supernatant containing cells that had detached from monolayers during incubation. Cells were washed with PBS and fixed in methanol and labeled using a mouse monoclonal antibody, anti-M30, according to the manufacturer's directions (Roche Molecular Biochemicals, Indianapolis, IN). Flow cytometry was performed on a FACScan using CELL-quest software (Becton Dickinson, Palo Alto, CA). Values are expressed as relative fluorescence index.
Cell Fractionation and Western Blot Analysis of Cytochrome c Release
T84 epithelial cells cultured in 60-mm plastic culture dishes were incubated with 0.1 U/ml leukocyte elastase for 0, 2, 4, and/or 12 h. Cells were washed once with PBS, scraped into PBS, sedimented at 1,000 g for 5 min and resuspended in hypotonic buffer [10 mM NaCl, 5 mM MgCl2, 10 mM Tris·HCl (pH 7.5), 100 µM PMSF]. Cells were allowed to swell on ice for 10 min and homogenized with a tight pestle using a 21-gauge needle (10 strokes) before lysis by the addition of isotonic homogenizing buffer [2.5x mannitol sucrose (MS) buffer, 525 mM mannitol, 175 mM sucrose, 12.5 mM Tris·HCl (pH 7.5), and 2.5 mM EDTA (pH 7.5)]. After being mixed, the suspension was centrifuged at 1,300 g for 5 min three times. Supernatants were collected and sedimented at 17,000 g for 15 min. Pellets were resuspended in 1x MS buffer and used as the heavy membrane fraction containing mitochondria. Supernatants were further sedimented at 100,000 g for 1 h, and the resultant supernatants were used as the cytosolic fraction.
SDS-PAGE and Immunoblotting
For SDS-PAGE of whole cell extracts, filter inserts were excised and solubilized directly into boiling lysis buffer (2% SDS, 10% glycerol, 65 mM Tris·HCl, pH 6.8, 50 mM dithiothreitol). Where indicated, cells were also collected from the bottom well and sedimented in microfuge tubes. The proteins were solubilized in boiling Laemli buffer and combined with whole cell extracts as described above. For Western blot analysis of cell fractions, samples were mixed directly with boiling Laemli sample buffer. After electrophoresis, proteins were transferred to nitrocellulose membranes, blocked with 5% low-fat milk in PBS with 0.05% Tween-20, and incubated overnight at 4°C with the relevant antibodies. Primary antibodies included anti-Akt (rabbit polyclonal); anti-phosphoSer-473 Akt (rabbit polyclonal); anti-phospho-GSK3/
(Ser21/9) (rabbit polyclonal); anti-caspase-3 (rabbit polyclonal); anti-cleaved caspase-6 (rabbit polyclonal); anti-caspase-8 (murine monoclonal); anti-caspase-9 (rabbit polyclonal); anti-cytochrome c; and anti-actin (murine monoclonal). Primary antibodies were from Cell Signaling Technology (Beverly, MA) with the exception of anti-actin antibody, which was from Valcant Pharmaceuticals, International (Costa Mesa, CA). After being washed, blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG at 1:5,000 dilution for 1 h at room temperature and developed by enhanced chemiluminescence system according to the manufacturer's instructions (Amersham Canada, Oakville, ON). When indicated, blots were stripped [10% SDS, 1 M Tris (pH 6.8), 0.8%
-mercaptoethanol at 50°for 20 min] and reprobed with relevant antibodies. In some instances, blots were reprobed with antibodies to
-actin to correct for any differences in protein loading. Quantitative densitometric analysis of the blots was done by using commercial image-analysis software (IP Lab Gel), and the intensity of all blots was within the linear range of the film.
Elastase Assay
Neutrophil elastase activity was determined on samples of lysed cells using EnzChek Elastase Assay Kit (Molecular Probes) according to the manufacturer's instructions.
Expression of Nitric Oxide Synthase mRNA
Total RNA was isolated from epithelial cells using TRIzol reagent (TRI; Sigma) according to the manufacturer's instructions. Purity was checked by A260/A280 ratio. Total RNA was reverse transcribed using random hexamer priming and SuperScript II RNAse H RT (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA). cDNA was prepared from 5 µg RNA (20-µl volume) by the addition of 50 U SuperScript II RT, 2 µl 10x RT buffer, 40 U RNaseOut recombinant ribonuclease inhibitor, 5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTP mix, and 7.5 ng/µl random hexamers. A negative control reaction lacking RT was performed for each RNA sample. The random hexamer primer was annealed for 10 min at 25°C. cDNA synthesis was performed for 50 min at 42°C, followed by a 15-min termination of the reactions at 70°C. RNAse H (1 µl) was added to each tube and incubated for 20 min at 37°C; cDNA was stored at 20°C. PCR was performed in 100 µl of reaction solution containing 0.2 mM dNTPs, 1.5 mM MgCl2, 2.5 U of Taq polymerase (Fermentas, Burlington, ON), and 0.5 µM oligonucleotide primers. Probes included human inducible nitric oxide (NO) synthase (iNOS; Accession No. L09210) sense 5'-CATTCAGATCCCCAAGCTCTACA-3', antisense 5'-GAGCCTCATGGTGAACACGTT-3', predicted PCR product 139 bp; human endothelial NOS (eNOS; Accession No. NM 000603) sense 5'-ACATTGAGAGCAAAGGGCTG-3', antisense 5'-CGGCTTGTCACCTCCTGG-3', predicted PCR product 425 bp; human GAPDH (Accession No. M33197) sense 5'-CAACGACCACTTTGTCAAGCTCA-3', antisense 5'-GCTGGTGGTCCAGGGGTCTTACT-3', PCR product 120 bp, and first-strand cDNA products. After an initial denaturation step (3 min at 94°C), PCR mixtures were amplified by 30 cycles (each including 30 s at 94°C, 30 s at 58°C, 30 s at 72°C). The final extension period was 5 min at 72°C. PCR products were analyzed in a 2% agarose gel and visualized using ultraviolet illumination.
NO Assay
T84 cells were exposed to control buffer (HBSS) or varying concentrations of elastase (0.1 U/ml or 0.3 U/ml) in the absence or presence of the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mM x 1 h) or L-N6-(1-iminoethyl)-lysine (L-NIL; 1.0 mM x 1 h); supernatant was collected at either 12 or 24 h and stored at 80°C until analyzed. NO was assayed using a colorimetric assay (Greiss assay; Calbiochem-Novobiochem) as per the manufacturer's instructions.
Statistical Analysis
Parametric data were compared by using t-tests for mean values or analysis of variance with correction for multiple comparisons (Fisher's portected least-significant difference test) when more than two variables were analyzed. Nonparametric data were compared using the Mann-Whitney U statistical method.
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RESULTS |
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To quantify the occurrence of epithelial cell apoptosis, neutrophils were induced to transmigrate across monolayers of T84 epithelial cells in the physiological basolateral to apical direction by a transepithelial gradient of fMLP (106 M), a potent neutrophil chemoattractant and activating agent. Monolayers were fixed and studied 18 h following the addition of 3 x 106 neutrophils, when most neutrophils were no longer present within the monolayer (24). The number of apoptotic epithelial cells was significantly increased following induced neutrophil transmigration as determined by nuclear morphology in monolayers stained with hematoxylin and eosin (control monolayers: 40 ± 2 apoptotic cells/10 HPF vs. neutrophil-treated monolayers: 123 ± 16 cells/10 HPF, P < 0.05 Mann-Whitney U-test, Fig. 1, A and B). As an independent method to confirm these observations, we assessed apoptosis using the TUNEL assay. These studies confirmed that there was a significant increase in apoptosis of epithelial cells following induced neutrophil transmigration (control monolayers: 5 ± 1 apoptotic cells/25 HPF vs. neutrophil treated monolayers: 29 ± 10/25 HPF, P < 0.05 Mann-Whitney U-test, Fig. 1, C and D). Additionally, apoptotic epithelial cells were present in the bottom chamber, having detached from the monolayer. A portion of these detached apoptotic epithelial cells was adherent to neutrophils (Fig. 1E). Apoptosis in detached epithelial cells was associated with alterations in epithelial mitochondrial membrane potential as determined using the fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbo-cyanine iodide (Desipher Assay; Fig. 1F), consistent with activation of the intrinsic apoptotic pathway in epithelial cells induced by neutrophil transmigration (vide infra).
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Elastase Induces Epithelial Cell Apoptosis
We have previously demonstrated (24) that leukocyte elastase contributes to alterations in epithelial barrier function. To determine whether leukocyte elastase contributes to the apoptosis in epithelial cells observed during the transmigration studies (Fig. 1), intact neutrophils were treated with a specific membrane-permeant elastase inhibitor, DMP-777 (24), before fMLP-induced neutrophil transmigration across epithelial monolayers. Pretreatment of neutrophils with DMP-777 completely inhibits leukocyte elastase activity (24). Under these conditions, there was no significant increase in the number of apoptotic epithelial cells following fMLP-induced neutrophil transmigration as determined by nuclear morphology in monolayers stained with hematoxylin and eosin (control monolayers in the absence of neutrophil transmigration: 40 ± 2 apoptotic cells/10 HPF; DMP-777-treated neutrophils applied to monolayers: 66 ± 5 cells/10 HPF; vehicle-treated neutrophils applied to monolayers: 123 ± 16 cells/10 HPF). These data implicate leukocyte elastase in the pathogenesis of epithelial apoptosis resulting from neutrophil transmigration and activation.
To further ascertain the effect of neutrophil elastase on epithelial monolayers, we used the single cell system in which neutrophil lysates were applied to epithelial monolayers. In these experiments, neutrophils were treated with a specific inhibitor of intracellular elastase DMP-777 (24) before their lysis and application to epithelial monolayers. Apoptosis in these experiments was assessed by flow cytometry using cytokeratin 18 cleavage. Exposure to lysates from neutrophils pretreated with DMP-777 resulted in a significantly smaller degree of apoptosis of epithelial cells at 18 h, compared with epithelial cells exposed to lysates from untreated neutrophils (14.8 ± 3.2% apoptotic cells with DMP-777-treated lysates vs. 23.9 ± 2.5 for untreated lysates, P < 0.05 ANOVA; Fig. 2, B and C). These data provide additional evidence that neutrophil elastase is, at least in part, responsible for the induction of apoptosis in epithelial cells.
To provide additional direct evidence that that the observed effects of activated neutrophils on epithelial apoptosis were attributable to leukocyte elastase, we incubated epithelial monolayers with purified leukocyte elastase. To approximate the concentration of elastase present during neutrophil transmigration in our model system, we first measured the activity of elastase in the supernatant of lysed neutrophils. These studies revealed that the supernatant from 10 x 106 cells yielded 0.1 ± 0.02 U/ml elastase activity. By comparison, the amount of elastase released from 10 x 106 neutrophils adhering to tissue culture plastic for 23 h in the presence of fMLP (106 M) was 0.035 ± 0.01 U/ml. We reasoned that neutrophils transmigrating across epithelial monolayers are in direct contact with adjacent epithelial cells, creating a microenvironment where the local concentration of elastase might be higher (40). Additionally, the release of higher levels of neutrophil elastase have been reported by other stimulants (20). Accordingly, we chose a concentration of 0.1 U/ml of elastase to pursue further studies to model the localized secretion by neutrophils into the interepithelial ("protected") space. Exposure of epithelial cells to 0.1 U of purified elastase for 18 h resulted in an enhanced rate of apoptosis compared with the basal apoptotic rates in control monolayers (elastase 0.1 U: 26.2 ± 2.9% apoptotic cells vs. control monolayers: 16.4 ± 2.5, P < 0.05 ANOVA; Fig. 3, A and B) as determined by cytokeratin cleavage assessed by flow cytometry.
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As an independent assessment of alterations of mitochondrial membrane permeability, we assessed cytochrome c release into the cytosol. In these experiments, epithelial monolayers were treated with purified leukocyte elastase and the amount of cytochrome c present in mitochondrial and cytosolic fractions of cells was assessed using Western blot analysis (Fig. 4). In control samples, cytochrome c was present only in the mitochondrial fraction. By contrast, in epithelial cells exposed to purified elastase, there was redistribution of cytochrome c from the mitochondrial to the cytosolic fraction within 4 h of elastase exposure. These studies provide direct support for the notion that leukocyte elastase induces alterations in epithelial mitochondrial membrane permeability. These data implicate leukocyte elastase as a mediator of epithelial cell apoptosis via activation of the intrinsic pathway.
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To further characterize the mechanisms by which leukocyte elastase induced apoptosis of epithelial cells, we sought to determine which caspase cascades are activated by elastase. For these experiments, epithelial cells were exposed to purified leukocyte elastase for varying times following which cells from the entire filter insert were lysed directly in identical volumes of denaturing SDS-containing lysis buffer. Cleavage products of caspase-9 were detected 6 h following the addition of purified elastase (Fig. 5A). Cleavage products of caspases-3, -6, and -8 were only detected at later time points (18 h) following the addition of purified elastase (Fig. 5, BD). These observations are consistent with the alterations in mitochondrial membrane potential described above and indicate that elastase exposure induces an initial alteration of epithelial mitochondrial membrane permeability with leakage of cytochrome c and subsequent activation of caspase-9 followed by activation of downstream caspases-3, -6, and -8. Although caspase-8 is more commonly thought of as the "apical" caspase in the extrinsic pathway, its activation as a downstream caspase also occurs in the intrinsic pathway.
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Akt is a serine/threonine kinase that is a major determinant of cell survival, an effect mediated, in part, through its ability to phosphorylate and inactivate the proapoptotic proteins BAD and procaspase-9 (9, 13, 14). To determine whether neutrophil transmigration modulated epithelial Akt, neutrophils were induced to transmigrate across epithelial monolayers (basolateral to apical direction) by a transepithelial gradient of fMLP. In initial experiments, we observed that our ability to assess alterations in epithelial Akt at early time points during neutrophil transmigration was complicated by the presence of two cell types, making it difficult to distinguish signals from neutrophils and epithelial cells. Accordingly, we focused on assessment of epithelial Akt activity at later time points when most neutrophils were no longer present within the monolayer. Eighteen hours after induction of neutrophil transmigration, epithelial cells from the filter were lysed directly in identical volumes of denaturing lysis buffer and analyzed by SDS PAGE and Western blot analysis using phospho-Akt specific antibodies. These experiments revealed that phosphorylation of epithelial Akt was decreased in epithelial monolayers subject to PMN transmigration compared with controls (Fig. 6, A and C).
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The use of purified elastase enabled us to examine alterations in Akt at earlier times. These studies demonstrated that there was a biphasic alteration in the phosphorylation of epithelial Akt with an initial increase peaking at 3 h after exposure to elastase (Figs. 7A and 8A) followed by a decrease below baseline control levels by 18 h (Figs. 6B and 7A). Increasing concentrations of elastase resulted in higher levels of Akt phosphorylation at early time points, an effect that reached a plateau at 0.1 U (Fig. 7B).
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Phosphorylation of Epithelial Akt and GSK3 are Dependent on the Activity of PI3-Kinase and Src Tyrosine Kinases
Akt activation is known to be dependent on PI3-kinase in other systems. To determine whether activation of Akt following elastase exposure is dependent on PI3-kinase activity, we pretreated epithelial cells with LY294002, a specific inhibitor of PI3-kinase, before incubation with elastase for 3 h. Samples were then subjected to SDS-PAGE and immunoblot analysis for Akt p473 and total Akt (Fig. 8, A and B). Pretreatment of epithelial cells with LY294002 inhibited phosphorylation of epithelial Akt following exposure to leukocyte elastase, compared with epithelial cells pretreated with vehicle alone (Fig. 8, A and B). Similarly, pretreatment with LY294002 inhibited elastase-induced phosphorylation of epithelial GSK3 (Fig. 8, C and D). Although several studies have reported that the activation of Akt can occur in a PI3-kinase-independent mechanism (54, 56, 67), these observations support the notion that elastase-induced activation of Akt at early time points is PI3-kinase dependent.
Recent studies (32) have demonstrated the tyrosine kinase Src can also regulate the activity of Akt. Evaluation of epithelial monolayers using Western blot analysis following exposure to leukocyte elastase showed increased levels of phosphorylation of Src at Y416 (stimulatory site) compared with control monolayers, whereas phosphorylation of Src at Y527 (inhibitory site) was unaffected (Fig. 8, E and F). To determine whether Src kinase activation is necessary for Akt activation, we pretreated epithelial cells with an Src family tyrosine kinase inhibitor (PP2) before incubating cells with elastase for 3 h. Samples were then subjected to SDS-PAGE and immunoblot analysis for Akt p473 and total Akt. This treatment resulted in inhibition of phosphorylation of epithelial Akt following exposure to leukocyte elastase compared with cells treated with vehicle control (Fig. 8, A and B). Similarly, pretreatment of cells with PP2 inhibited, to a lesser extent, phosphorylation of epithelial GSK3 following exposure to leukocyte elastase, compared with epithelial cells treated with elastase alone (Fig. 8, C and D). In concert, these data support the notion that elastase-induced activation of Akt is mediated by Src-family tyrosine kinases (11)
Inhibition Of Epithelial Akt Increases Elastase-Mediated Apoptosis
To determine whether activation of Akt influenced epithelial survival following elastase exposure, epithelial cells were pretreated with a specific inhibitor of Akt before incubation with purified elastase. There was no significant difference in the basal rates of apoptosis of the epithelial cells compared with control cells treated with vehicle alone in the absence of elastase exposure. However, pretreatment of epithelial cells with the Akt inhibitor resulted in a significant increase in apoptosis in response to subsequent exposure to elastase compared with epithelial cells exposed for 18 h to leukocyte elastase alone (Akt inhibitor + elastase 0.1 U: 40.6 ± 5.8% apoptotic cells vs. elastase 0.1 U alone: 26.2 ± 2.9, P < 0.01 ANOVA; Fig. 3, CE). The role of PI3-kinase as an upstream activator of Akt in this pathway could not be ascertained because incubation of epithelial cells with LY294002 alone resulted in significantly increased levels of epithelial apoptosis as measured by flow cytometry using the M30 antibody (data not shown).
Because NOS is regulated by Akt under certain circumstances, we evaluated whether epithelial production of NO was altered by exposure to elastase. Using RT-PCR, we first ascertained that epithelial cells expressed both eNOS and iNOS (data not shown). However, epithelial NO production was not altered following exposure to elastase as determined by the Greiss assay (data not shown). Moreover, inhibition of NOS with either L-NAME or L-NIL did not prevent elastase-induced apoptosis (data not shown). Taken together with the previous data, these observations are consistent with the notion that exposure of epithelial cells to leukocyte elastase induces an initial PI3-kinase and Src-dependent activation of Akt ("survival pathway") that is independent of NO production by epithelia.
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DISCUSSION |
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Our current study supports and extends published studies. Recently, an in vitro study suggested that neutrophil transmigration is associated with increased apoptosis of T84 epithelial cells in a Fas ligand-independent manner. Decreased levels of total caspase-9 followed by decreased caspase-3 were observed (42). In the current manuscript, we provide further evidence that epithelial apoptosis during acute inflammation may result from alterations in epithelial mitochondrial membrane permeability and activation of the intrinsic apoptotic pathway and provide evidence that leukocyte elastase is partly responsible for induction of epithelial apoptosis during inflammation.
The effects of elastase in vivo and in vitro are complex. Elastase has diverse potential substrates and can exert both pro- and anti-inflammatory effects (40). The importance of leukocyte elastase in acute inflammatory epithelial injury is supported by reports of increased levels of elastase in the intestinal lumen of patients with inflammatory bowel disease (2, 28) and in bronchoalveolar lavage fluids from patients with acute respiratory distress syndrome and cystic fibrosis (40). Attenuation of intestinal damage occurs in animal models of acute colitis and cystic fibrosis following administration of antielastase compounds (7, 46). Interest in the role of elastase in lung injury was sparked by the observation that unchecked elastase activity, due to a genetic defect in an inhibitor of elastase activity (1-antitrypsin deficiency), resulted in an early and sometimes severe form of emphysema (18). Further studies in animal models demonstrated that the intratracheal administration of elastase is associated with goblet cell hyperplasia and increased secretion of mucins and apoptosis of lung epithelial cells (43), supporting a nondegradative role for elastase (34, 44). Other studies (38, 55) focused on animal models of lung injury demonstrated attenuation of injury with the administration of elastase inhibitors. However, mechanisms by which leukocyte elastase induces cellular hyperplasia compared with injury of respiratory and gastrointestinal epithelium and how this contributes to the inflammatory process remain unclear.
Earlier work (66) evaluating the effects of elastase on endothelial cells showed an increase in endothelial apoptosis. In the current study, we demonstrated that epithelial cell apoptosis is increased by exposure to leukocyte elastase. Initiation of epithelial apoptosis by leukocyte elastase may occur via alterations in the mitochondrial membrane permeability and activation of the intrinsic apoptotic cascade. These conclusions are supported by several lines of evidence. For example, epithelial exposure to elastase resulted in the redistribution of cytochrome c from the mitochondria to the cytosol of epithelial cells, indicating increased mitochondrial permeability. In addition, cleavage of caspase-9 was detected before cleavage of caspase-3. Both of these observations are consistent with alterations in mitochondrial permeability as an initiating event in epithelial apoptosis. Although the activation of caspase-8 may be a consequence of the activation of the upstream caspases-9, -3, and -6, we cannot exclude the possibility that it results from trimerization of Fas receptors and parallel activation of the extrinsic apoptotic. In this regard, anoikis has been associated with overexpression of Fas ligand (53). Reports that epithelial cells can release TNF- following hypoxic injury (60) raise the possibility that elastase upregulates production of TNF-
in an analogous manner, resulting in induction of apoptosis through the extrinsic pathway. Alternately, trimerization of Fas receptors may occur independently of ligand binding in several conditions including cell shrinkage (22), which can occur in association with cell detachment (61). Further studies are needed to distinguish between these possible mechanisms.
Activation of caspase cascades can be further regulated by proteins within the cell such as the survival protein Akt. Active Akt can downregulate apoptosis initiated by alterations in mitochondrial permeability by several mechanisms including inactivation of the proapoptotic protein BAD or inhibition of procaspase-9 cleavage into its active form. Decreased Akt activity has been reported in anoikis (3), and inhibition of Akt increases cell susceptibility to Fas-mediated apoptosis (1). Recent studies have demonstrated that there is activation of PI3-kinase following integrin engagement (23) and, conversely, that there is decreased Akt activity following disruption of cell anchorage (39). We observed decreased levels of phosphorylated epithelial Akt following neutrophil transmigration across epithelia as well as following epithelial incubation with leukocyte elastase for 18 h. These decreased levels may be partly attributable to disruption in cell adhesion. Interestingly, when we measured Akt phosphorylation at early time points after exposure to elastase, we observed it to be increased. This increase was dependent on PI3-kinase and Src tyrosine kinase. PI3-kinase activation of Akt is well established (for a review, see Ref. 8), whereas Src has only recently been shown to interact with the COOH-terminal regulatory region of Akt, where it is responsible for Akt activation by growth factors (32).
Specific inhibition of epithelial Akt resulted in even greater rates of epithelial cell apoptosis in response to leukocyte elastase. One interpretation of these data is that epithelial cells initially activate Akt in response to the stress of elastase exposure in a PI3-kinase-dependent fashion. This activation partly protects epithelial cells from the proapoptotic effects of leukocyte elastase. This effect, however, is transient as evidenced by the late decreased levels of phosphorylated Akt, perhaps representing a state in which the cellular survival signals are overwhelmed. These data are consistent with the activation of both pro- and antiapoptotic signaling pathway response to epithelial exposure to leukocyte elastase, with activation of survival pathways moderating cellular injury mediated by the initiation of apoptotic pathways.
Akt has been shown to have a role in modulating numerous intracellular processes. In addition to inhibiting apoptosis by phosphorylating BAD and preventing procaspase-9 cleavage, it is well known to regulate transcription by inactivating GSK3 (13, 14). More recently, Akt has been implicated in the regulation of the endothelial (21) and inducible isoforms of NOS in macrophages (47, 59). In this regard, NO has been reported to be both beneficial (49) and harmful (12) for cell survival. In our system, we demonstrated that T84 epithelial cells express both eNOS and iNOS but were unable to detect altered levels of NO in response to elastase exposure. An alternate mechanism may be that activation of Akt delays procaspase-9 cleavage, thereby attenuating the proapoptotic effects of leukocyte elastase. Future studies should focus on mechanisms by which alterations in Akt activity modulate cell survival during inflammatory injury.
In summary, epithelial apoptosis is increased following exposure to leukocyte elastase, resulting in increased mitochondrial membrane permeability and downstream activation of the caspase cascade consistent with activation of the intrinsic, mitochondrial-dependent apoptotic pathway as the primary mechanism leading to apoptosis. Concomitant activation of Akt appears to have a moderating role in the early phases of this process. We suggest that there is a dual cellular response to elastase in acute inflammation that includes the activation of both proapoptotic and survival pathways, the balance of which ultimately determines the fate of the epithelial cell.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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