Fas (CD95) Induces Alveolar Epithelial Cell Apoptosis in Vivo
2001; Elsevier BV; Volume: 158; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63953-3
ISSN1525-2191
AutoresGustavo Matute‐Bello, Robert K. Winn, Mechthild Jonas, Y. Emil, Thomas R. Martin, W. Conrad Liles,
Tópico(s)Blood disorders and treatments
ResumoActivation of the Fas/FasL system induces apoptosis of susceptible cells, but may also lead to nuclear factor κB activation. Our goal was to determine whether local Fas activation produces acute lung injury by inducing alveolar epithelial cell apoptosis and by generating local inflammatory responses. Normal mice (C57BL/6) and mice deficient in Fas (lpr) were treated by intranasal instillation of the Fas-activating monoclonal antibody (mAb) Jo2 or an irrelevant control mAb, and studied 6 or 24 hours later using bronchoalveolar lavage (BAL), histopathology, DNA nick-end-labeling assays, and electron microscopy. Normal mice treated with mAb Jo2 had significant increases in BAL protein at 6 hours, and BAL neutrophils at 24 hours, as compared to lpr mice and to mice treated with the irrelevant mAb. Neutrophil recruitment was preceded by increased mRNA expression for tumor necrosis factor-α, macrophage inflammatory protein-1α, macrophage inflammatory protein-2, macrophage chemotactic protein-1, and interleukin-6, but not interferon-γ, transforming growth factor-β, RANTES, eotaxin, or IP-10. Lung sections from Jo2-treated normal mice showed neutrophilic infiltrates, alveolar septal thickening, hemorrhage, and terminal dUTP nick-end-labeling-positive cells in the alveolar septae and airspaces. Type II pneumocyte apoptosis was confirmed by electron microscopy. Fas activation in vivo results in acute alveolar epithelial injury and lung inflammation, and may be important in the pathogenesis of acute lung injury. Activation of the Fas/FasL system induces apoptosis of susceptible cells, but may also lead to nuclear factor κB activation. Our goal was to determine whether local Fas activation produces acute lung injury by inducing alveolar epithelial cell apoptosis and by generating local inflammatory responses. Normal mice (C57BL/6) and mice deficient in Fas (lpr) were treated by intranasal instillation of the Fas-activating monoclonal antibody (mAb) Jo2 or an irrelevant control mAb, and studied 6 or 24 hours later using bronchoalveolar lavage (BAL), histopathology, DNA nick-end-labeling assays, and electron microscopy. Normal mice treated with mAb Jo2 had significant increases in BAL protein at 6 hours, and BAL neutrophils at 24 hours, as compared to lpr mice and to mice treated with the irrelevant mAb. Neutrophil recruitment was preceded by increased mRNA expression for tumor necrosis factor-α, macrophage inflammatory protein-1α, macrophage inflammatory protein-2, macrophage chemotactic protein-1, and interleukin-6, but not interferon-γ, transforming growth factor-β, RANTES, eotaxin, or IP-10. Lung sections from Jo2-treated normal mice showed neutrophilic infiltrates, alveolar septal thickening, hemorrhage, and terminal dUTP nick-end-labeling-positive cells in the alveolar septae and airspaces. Type II pneumocyte apoptosis was confirmed by electron microscopy. Fas activation in vivo results in acute alveolar epithelial injury and lung inflammation, and may be important in the pathogenesis of acute lung injury. 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Although it has been reported that repeated activation of Fas in the lungs of mice results in pulmonary fibrosis after 2 weeks,32Hagimoto N Kuwano K Miyazaki H Kunitake R Fujita M Kawasaki M Kaneko Y Hara N Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen.Am J Respir Cell Mol Biol. 1997; 17: 272-278Crossref PubMed Scopus (258) Google Scholar the acute effects of Fas activation in the lungs are unclear. We hypothesized that sFasL in the airspaces may contribute to the pathogenesis of acute lung injury by inducing apoptosis of epithelial cells, as well as by stimulating the release of inflammatory cytokines via nuclear factor κB activation. The major goal of this study was to determine whether activation of Fas in the alveoli of the lungs would induce apoptosis of type II pneumocytes in the alveolar wall, and initiate an inflammatory response in the lungs. We treated normal mice or naturally occurring mutant mice deficient in Fas expression (lpr mice) with the monoclonal antibody (mAb) Jo2, which activates Fas on the surface of cells in vitro and in vivo.32Hagimoto N Kuwano K Miyazaki H Kunitake R Fujita M Kawasaki M Kaneko Y Hara N Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen.Am J Respir Cell Mol Biol. 1997; 17: 272-278Crossref PubMed Scopus (258) Google Scholar, 33De Leon M Jackson KM Cavanaugh JR Mbangkollo D Verret CR Arrest of the cell cycle reduces susceptibility of target cells to perform-mediated lysis.J Cell Biochem. 1998; 69: 425-435Crossref PubMed Scopus (4) Google Scholar We identified apoptotic cells in the alveolar walls by DNA nick-end-labeling and electron microscopy, and the alveolar inflammatory response by quantitative histology, BAL, and cytokine ribonuclease protection assays (RPA). The activating anti-Fas mAb Jo2 (Armenian hamster IgG) and a control mAb (Armenian hamster anti-TNP IgG) were purchased from PharMingen (San Diego, CA). Both antibodies were free of azide and endotoxin, as determined by the manufacturer. In addition, the antibodies were confirmed to contain <0.01 endotoxin U/ml by the limulus amebocyte assay (ECL-1000; Biowhittaker, Walkersville, MD). Male mice, weighing 20 to 30 g, were briefly anesthetized with inhaled halothane. Either mAb Jo2 or control mAb at a dose of 2.5 μg/g was administered to each mouse by intranasal instillation in a solution containing 1 mg/ml of mAb in sterile phosphate-buffered saline (PBS), as previously described.34Szarka RJ Wang N Gordon L Nation PN Smith RH A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation.J Immunol Methods. 1997; 202: 49-57Crossref PubMed Scopus (179) Google Scholar The animals were allowed to recover from anesthesia, returned to their cages, and given free access to water and food. At the end of the experiment the animals were euthanized with ketamine and xylazine, the thorax was rapidly opened and the animal was exsanguinated by direct cardiac puncture. The lungs were dissected free and the trachea was cannulated to perform BAL, or to fix the lungs, or to extract mRNA. The protocol was approved by the animal care committee of the University of Washington. We used male mice weighing 20 to 30 g. The mice were either C57BL/6 mice, (B&K Universal, Seattle, WA), or naturally occurring mutant mice lacking the Fas receptor (lpr mice) (Jackson Laboratory, Bar Harbor, ME). The lpr mice are derived from the C57BL/6 mouse strain. The mice were treated with either the Fas-activating mAb Jo2 or an irrelevant mAb (hamster anti-TNP IgG) as described above, and euthanized at either 6 or 24 hours after the administration of the antibody. Studies with lpr mice were performed to confirm that effects observed with mAb Jo2 in C57BL/6 mice were specific for Fas activation. As an additional comparison, C57BL/6 were treated with an irrelevant mAb (hamster anti-TNP IgG) and euthanized at either 6 or 24 hours. BAL was performed by instilling 0.9% NaCl containing 0.6 mmol/L ethylenediaminetetraacetic acid in two separate 0.5 ml aliquots. The fluid was recovered by gentle suction and placed on ice for immediate processing. An aliquot of the BAL fluid was processed immediately for total and differential cell counts. Total cell counts were performed with a hemocytometer, whereas differential cell counts were performed on cytospin preparations stained with modified Wright-Giemsa stain (Diff-Quik; American Scientific Products, McGaw Park, IL). The remainder of the lavage fluid was spun at 200 × g for 30 minutes, and the supernatant was removed aseptically and stored in individual aliquots at −70°C. The total protein concentration in BAL fluid was measured using the bicinchoninic acid method (BCA assay; Pierce Co., Rockford, IL). Lung cytokine mRNA expression (RANTES, eotaxin, MIP-1α, MIP-2, IP-10, macrophage chemotactic protein-1 (MCP-1), tumor necrosis factor-α, interleukin-6, interferon-γ, transforming growth factor-β) was measured by RPA. Three mice were treated by intranasal instillation of mAb Jo2 (2.5 μg/g), and three control mice were treated with an irrelevant mAb. After 6 hours, the mice were euthanized and their lungs excised. Lung RNA was extracted with Triazol (Biotecx Laboratories, Inc., Houston, TX) according to the vendor’s instructions. RPA was performed using an RPAII kit (Ambion Inc., Austin, TX), with MCK-3 and MCK-5 template sets (Pharmingen, San Diego, CA) and [α-32P]UTP according to the manufacturer’s instructions. Samples were run under denaturing electrophoresis on 5% polyacrylamide gel, then imaged and analyzed in a Packard Cyclone Phosphorimager (Amersham Pharmacia Biotech, Piscataway, NJ). To control for relative differences in RNA loading between samples, the specific cytokine mRNA signals were normalized to the intensity of the respective glyceraldehyde-3-phosphate dehydrogenase signal. Results are expressed as relative mRNA expression (mean ± SEM), using the following equation: normalized Jo2 induced expression (n = 3)/normalized control expression (n = 3). The lungs were fixed by inflation with 10% neutral-buffered formalin at a transpulmonary pressure of 15 cm H2O and embedded in paraffin. Within 24 hours of fixation, lung sections were stained with hematoxylin and eosin for light microscopy, or by the DNA nick-end-labeling assay to evaluate apoptotic cells, or processed for transmission electron microscopy as described below. The slides were submerged in 10% neutral-buffered formalin for 10 minutes, followed by 70% ethanol for 5 minutes. The slides were rehydrated for 10 minutes in PBS and treated with 0.002% proteinase K (Sigma, St. Louis, MO.) in double-distilled water for 5 to 15 minutes at room temperature. Endogenous peroxidase was quenched by placing the slides in 2% hydrogen peroxide for 5 minutes. For equilibration, the slides were treated in Klenow labeling buffer (TACS In situ Apoptosis Detection Kit; Trevigen Inc., Gaithersburg, MD) for at least 1 minute and then incubated for 60 minutes at 37°C with Klenow enzyme and Klenow dNTP mix in Klenow labeling buffer (all reagents from Trevigen, Inc.) prepared according to instructions from the manufacturer. Negative control slides were incubated with the labeling mixture without the Klenow enzyme. After incubation the slides were completely submerged in Klenow Stop buffer (Trevigen Inc.) for 5 minutes at room temperature and rinsed in PBS for 2 minutes. The samples were then treated for 15 minutes with streptavidin-horseradish peroxidase detection solution (Trevigen Inc.), washed twice for 2 minutes in PBS and incubated in diaminobenzidine (Trevigen Inc.) for 7 minutes at room temperature. The samples were then rinsed twice in distilled water and stained with 1% methyl green in 0.1 mol/L sodium acetate (pH 4.0) for 5 minutes, quickly dehydrated in 95% and 100% ethanol, cleared in xylene, and mounted with Permount (Fisher Scientific, Pittsburgh, PA). Lung tissue was fixed for electron microscopy by immersion in 6.25% glutaraldehyde, 2% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer for 2 hours. The tissue was postfixed in 2% potassium-ferrocyanide in distilled water for 4 hours at room temperature, rinsed with distilled water, stained with 0.5% uranyl acetate for 20 minutes, and then rinsed in distilled water. The samples were dehydrated in a graded series of ethanol solutions and embedded in Eponate 12 (Ted Pella Inc., Redding, CA). Thin sections were cut from two randomly selected blocks with a diamond knife using an LKB Nova ultramicrotome and collected on parlodion-coated 200 mesh copper grids (Electron Microscopy Sciences, Fort Washington, PA.). The sections were stained with uranyl acetate and lead citrate and examined with a JEOL TEM 1200 EX at a magnification of ×3000 or higher. Only cells with a recognizable nucleus were included in the analysis. For each sample several sections from at least two electron microscopic blocks were used for evaluation. Each slide was evaluated by two separate investigators (GMB and WCL) in a blinded manner. To generate the lung injury score, a total of 300 alveoli were counted on each slide at ×400 magnification. Within each field, points were assigned according to predetermined criteria (Table 1). All of the points for each category were added and weighted according to their relative importance. The injury score was calculated according to the following formula: injury score = [(alveolar hemorrhage points/no. of fields) + 2 × (alveolar infiltrate points/no. of fields) + 3 × (fibrin points/no. of fields) + (alveolar septal congestion/no. of fields)]/total number of alveoli counted.Table 1Quantitative Histopathology Score of Lung InjuryTissue0123Alveolar septaeAll septae are thin and delicateCongested alveolar septae in less than 1/3 of the fieldCongested alveolar septae in 1/3 to 2/3 of the fieldCongested alveolar septae in greater than 2/3 of the fieldAlveolar hemorrhageNo hemorrhageAt least 5 erythrocytes per alveolus in 1 to 5 alveoliAt least 5 erythrocytes per alveolus in 5 to 10 alveoliAt least 5 erythrocytes per alveolus in more than 10 alveoliIntra-alveolar fibrinNo intra-alveolar fibrinFibrin strands in less than 1/3 of the fieldFibrin strands in 1/3 to 2/3 of the fieldFibrin strands in greater than 2/3 of the fieldIntra-alveolar infiltratesLess than 5 intra-alveolar cells per field5 to 10 intra-alveolar cells per field10 to 20 intra-alveolar cells per fieldMore than 20 intra-alveolar cells per field Open table in a new tab Comparisons between two groups were made with the two-tailed Fisher’s exact t-test. Comparisons between multiple groups were made with the Kruskall-Wallis analysis of variance and with factorial analysis of variance.35Rosner B Fundamentals of Biostatistics. Duxbury Press, Boston1982Google Scholar For post hoc analysis, the Fisher’s test was used. A P value <0.05 was considered significant. Six hours after intranasal instillation of mAb Jo2, the BAL total protein concentration was significantly increased in C57BL/6 mice (n = 5) as compared to the lpr mice (n = 5), or to the C57BL/6 mice treated with an irrelevant control mAb (n = 5) (Figure 1A). At 24 hours, all animal groups had similar concentrations of total protein in the BAL fluid (Figure 1B). In contrast to the early increase in BAL fluid total protein, the BAL fluid neutrophil response was delayed. Six hours after Jo2 instillation, there was trend toward more BAL fluid PMN in the C57BL/6 mice treated with the mAb Jo2, but this did not reach statistical significance (Figure 2A). At 24 hours, the number of BAL fluid PMN was significantly higher in C57BL/6 mice treated with mAb Jo2 (n = 5), as compared to the lpr group (n = 5), and to C57BL/6 animals treated with the control mAb (n = 5) (Figure 2B). To assess cytokine message expression we performed RPAs on mRNA extracted from the lungs of C57BL/6 mice 6 hours after instillation of either Jo2 mAb (n = 3) or control mAb (n = 3) (Figure 3). The cytokine expression was normalized to the GADPH signal to control for loading differences. Jo2-treated mice showed a sixfold increase in MIP-2 mRNA, a twofold increase in MIP-1α mRNA, a fourfold increase in MCP-1 mRNA, a threefold increase in tumor necrosis factor-α mRNA, and a 60-fold increase in interleukin-6 mRNA. In contrast, there was no increase in message for transforming growth factor-β, eotaxin, or IP-10. The relative expression of RANTES and interferon-γ were significantly decreased. At 24 hours, C57BL/6 mice treated with mAb Jo2 (n = 6) had a significantly higher lung injury score than animals treated with the control mAb (n = 7; P < 0.01) (Figure 4). The lung injury was characterized by patchy areas of neutrophilic infiltrates with thickening of the alveolar septae and areas of hemorrhage (Figure 5a). The majority of the cells infiltrating the airspaces, as well as cells in the alveolar septae, showed densely condensed nuclei suggesting apoptosis in areas of injury (Figure 5b). No histopathological abnormalities were present in animals that were treated with the control mAb (Figure 6, A and b).Figure 5H&E preparation and DNA nick-end-labeling assay from consecutive sections of the caudal lung lobe of a wild-type mouse 24 hours after intranasal instillation of Fas activating mAb Jo2. The H&E preparation shows inflammatory infiltrates and areas of hemorrhage [original magnifications: ×100 (a); ×400 (b)]. The DNA nick-end-labeling assay shows positive cells in the inflammatory exudate [original magnification, ×100 (c)] and in the alveolar walls (arrows) [original magnification, ×400 (d)]. Negative controls for the DNA nick-end-labeling assay are shown ine and f.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6H&E preparation and DNA end-nick-labeling assay from consecutive sections of the caudal lung lobe of a wild-type mouse 24 hours after intranasal instillation of an irrelevant control mAb. The H&E preparation shows normal lung architecture [original magnifications, ×100 (a); ×400 (b)], whereas the DNA nick-end-labeling assay shows no positive cells [original magnifications, ×100 (c); × 160 (d)]. Negative controls for the DNA nick-end-labeling assay are shown ine (original magnification, ×100) and f (original magnification, ×160).View Large Image Figure ViewerDownload Hi-res image Download (PPT) DNA nick-end-labeling assays confirmed the presence of apoptosis in cells infiltrating the airspaces (Figure 5, c and d). In addition, positive cells were present in the alveolar septae of areas of injury. No positive cells were seen in the animals treated with the control mAb (Figure 6, c and d). By electron microscopy, the lungs of a mouse treated with mAb Jo2 showed features characteristic of apoptosis in alveolar type II cells. These features included increased electron density of the cytoplasm, condensation of the chromatin, and vacuolization of the nuclear envelope (Figure 7).36Anglade P Tsuji S Vyas S Morphological diversity of programmed cell death: deciphering of triggering signals needs comprehensive classification.Biomed Res. 1997; 18: 1-6Google Scholar, 37Kerr JFR Gobe GC Winterford CM Harmon BV Anatomical methods in cell death.in: Schwartz LM Osborne BA Methods in Cell Biology. Academic Press, San Diego, CA1995: 2-27Google Scholar, 38Lockshin RA Zakeri Z The biology of cell death and its relationship to aging.in: Holbrook NJ Martin GR Lockshin RA Modern Cell Biology. Wiley-Liss, New York1996: 167-180Google Scholar Interestingly, there were also mitochondrial abnormalities. Changes consistent with apoptosis were not seen in any of the endothelial cells that were identified. The primary goal of this study was to determine whether activation of the Fas system in vivo results in damage to cells in the alveolar walls, and whether this event initiates an acute inflammatory response. We found that C57BL/6 mice developed patchy neutrophilic infiltrates, areas of alveolar hemorrhage, thickening of the alveolar septae, and apoptosis of type II pneumocytes within 24 hours after treatment with Fas-activating mAb Jo2. These histopathological changes were associated with an early increase in BAL fluid protein and early induction of cytokine gene expression, followed by a later increase in BAL fluid neutrophils. Similar changes did not occur in mice lacking Fas (lpr), or in mice treated with an irrelevant antibody. These findings provide clear evidence that the induction of apoptosis in cells of the alveolar wall initiates a sustained inflammatory response in the lungs. In humans, acute lung injury is characterized by epithelial and endothelial injury, neutrophilic alveolitis, and hyaline membrane formation. The primary event leading to lung injury in ARDS has not been established. A prevalent hypothesis is that the neutrophil mediates lung injury in ARDS.39Boxer LA Axtell R Suchard S The role of the neutrophil in inflammatory diseases of the lung.Blood Cells. 1990; 16: 25-42PubMed Google Scholar In this paradigm, uncontrolled neutrophil activation leads to the accumulation of oxidants and proteases in the lungs, which cause damage to the cells of the alveolar environment. However, neutrophils can migrate into the lungs of humans without causing injury, and large numbers of neutrophils can migrate into the lungs of sheep without causing injury to the tight epithelial barrier.40Wiener-Kronish JP Albertine KH Matthay MA Differential responses of the endothelial and epithelial barriers of the lung in s
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