Fas activation alters tight junction proteins in acute lung injury.

Abstract

The acute respiratory distress syndrome (ARDS) is characterized by protein-rich edema in the alveolar spaces, a feature in which Fas-mediated apoptosis of the alveolar epithelium has been involved. The goal of the present study was to determine whether Fas activation increases protein permeability by mechanisms involving disruption of the paracellular tight junction (TJ) proteins in the pulmonary alveoli. We show that activation of the Fas pathway increased protein permeability in mouse lungs and altered the expression of the TJ proteins occludin and ZO1 in the alveolar-capillary membrane in vivo and in human alveolar epithelial cell monolayers in vitro. Blockade of caspase-3, but not inhibition of tyrosine kinase dependent pathways, prevented the alterations in TJ protein expression and permeability induced by the Fas/FasL system in human alveolar cell monolayers in vitro. We also observed that both the increase of protein permeability and the disruption of TJ proteins induced by Fas, occurred before cell death could be detected in the cell monolayers in vitro. Thus, targeting caspase pathways could prevent the disruption of TJs and reduce the formation of lung edema in the early stages of ARDS.

INTRODUCTION

Accumulation of protein-rich edema fluid into the alveoli and interstitium occurs in the early stage of the acute respiratory distress syndrome (ARDS), due to an increase in protein permeability of the alveolar-capillary membrane and to an impairment of the alveolar fluid clearance capability of the alveolar epithelium (1). The alveolar epithelial barrier is considered a major player in the formation and resolution of this edema (2, 3).

The permeability and the alveolar fluid clearance capability of the alveolar epithelium are regulated by tight junctions (TJs), which are intercellular structures that prevent the passage of molecules through the paracellular spaces (4–6). Also, they maintain cellular polarity and regulate transcellular transport and a variety of intracellular signals (7, 8). They are composed of transmembrane proteins such as occludins, claudins, tricellulin and other junction adhesion molecules (JAM), as well as cytoplasmic proteins such as zonula occludens (ZO)-1, ZO-2, ZO-3 that ultimately bind to the actin fibers of the cytoskeleton (4–6). Preclinical studies have shown that TJs can be directly altered by various insults such as mechanical stress, viral and bacterial pathogens, or their products (e.g. endotoxin) (9–12). TJ proteins can also be altered by the activation of different cell signaling pathways, including caspases and protein kinases (13–20). However, the exact role of TJs on the disruption of the alveolar epithelial barrier has not been fully elucidated in ARDS.

Activation of the receptor protein Fas by its natural ligand, FasL, is considered to play an important role in the development of ARDS (21–25). Our previous studies showed that activation of the Fas/FasL system increased alveolar-capillary protein permeability and impaired alveolar fluid clearance in mice, leading to lung edema formation, by mechanisms involving caspase-mediated apoptosis in the alveolar walls (23). In addition, activation of the Fas/FasL system induces inflammatory responses in the lung, including cytokine release from epithelial cells via activation of protein kinases (24, 26–28). In most models of acute lung injury, however, the number of apoptotic cells in the alveolar walls is relatively small. Although this situation could be explained by a rapid clearance of apoptotic cells, it is conceivable that pulmonary edema mediated by Fas/FasL may not be exclusively due to the ultimate death of alveolar epithelial cells. We hypothesized that Fas activation leads to lung edema formation by altering the structure of TJs without requiring the ultimate death of the cells.

In the present study, we determined the effect of human soluble FasL on pulmonary TJ proteins in mouse lungs in vivo and in human primary alveolar epithelial cell monolayers in vitro. We found that Fas activation by human soluble FasL increased the permeability and altered the expression and distribution of the TJ proteins occludin and ZO1 in alveolar epithelial cells. Such changes occurred prior to cell death and were initially mediated by the activation of the apoptotic precursor caspase-3, but not by pro-inflammatory tyrosine kinase pathways.

MATERIAL AND METHODS

Reagents

The long form of recombinant human soluble FasL (rh-sFasL) from ENZO Life Science (San Diego, CA) was used for in vivo studies. For in vitro experiments, we used a long form of rh-sFasL (103–281 aa, containing the stalk region) and a short form (134–281 aa, non-containing the stalk region) from Abcam, as indicated in each experiment. For immunoblotting, we used the following reagents: monoclonal mouse anti-Occludin (clone OC-3F10) (Invitrogen), polyclonal rabbit anti-ZO1 (Invitrogen), Alexa Fluor®546 conjugated-goat anti-mouse IgG (H+L) antibody, (ThermoFisher Scientific), Alexa Fluor®568 conjugated-goat anti-Rabbit IgG (H+L) (ThermoFisher Scientific), 4 ‘,6-diamino-2-fenilindol (DAPI) from Invitrogen. Proteinase K, protein block serum-free, fluorescent mounting medium and microscope slides were obtained from DAKO. In situ cell death detection kit-fluorescein for the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) technique was purchased from Roche. The irreversible caspase-3 inhibitor zDEVD.FMK, and the negative control for caspase inhibitors zFA.FMK were purchased from BD Pharmingen. The tyrosine kinase inhibitor genistein was obtained from Sigma-Aldrich. The universal tyrosine kinase assay kit was obtained from Takara-Clontech. Fluorescein (FITC)-human serum albumin protein (full length) was purchased from Abcam. Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich.

Animal studies

The animal protocols were approved by the Animal Care Committee of Hospital Universitario de Getafe (Madrid, Spain). Male C57BL/6 mice or naturally occurring mutant mice lacking functional Fas receptor (B6.MRL-Fas lpr/J mice) (lpr mice) (The Jackson Laboratory, Bar Harbor, ME) weighing 25–30 g were anesthetized with inhaled 2–5% isofluorane and treated once by intratracheal instillation of recombinant human soluble FasL (rh-FasL, 25 ng/g) or PBS. The number of mice used per group was 10. For the histology analyses, 5 additional mice were included in the study. This dose of rh-FasL was chosen from a previous dose-response experiment showing that 25 ng/g of FasL was the minimal dose causing the highest levels of neutrophil recruitment into the alveolar airspaces (data not shown). After the instillations, the mice were allowed to recover from anesthesia and returned to their cages with free access to food and water. The mice were euthanized at 16 h after instillation with an intraperitoneal injection of pentobarbital (120 mg/kg) and exsanguinated by closed cardiac puncture. The thorax was opened rapidly, the trachea was cannulated with a 20-gauge catheter, the left hilum was clamped, and the left lung was removed, rapidly weighed on a precision balance for evaluating the left lung weight-to-body weight ratio, and flash-frozen in liquid nitrogen for protein and enzyme analyses. The right lung was either fixed by intratracheal instillation of 4% paraformaldehyde at a transpulmonary pressure of 15 cm of water and then embedded in paraffin for histology analysis or instilled with five separate 0.5-ml aliquots of 0.9% NaCl containing 0.6 mM EDTA at 37°C to obtain the bronchoalveolar lavage (BAL) fluid.

Analysis of mouse bronchoalveolar lavage fluid

The bronchoalveolar lavage (BAL) fluid samples from the right lung were processed immediately for total and differential cell counts. Total white cell counts were performed with a hemocytometer, and differential counts were performed by using Advia®2120i System analyzer. The remainder of the lavage fluid was spun at 200 g for 30 min, and the supernatant was removed aseptically and stored in individual aliquots at −80°C. The concentration of IgM in BAL fluid was measured by ELISA (Bethyl Laboratories, Montgomery,TX) following the manufacturer’s instructions. The lower limit of detection of the IgM assay was 20 ng/ml.

Histological methods in mouse lung tissue

Paraffin-embedded murine lung tissue sections (4 μm thick) were obtained. TUNEL fluorescent staining for detection of DNA damage in situ was performed according to the manufacturer’s instructions (Roche Diagnostics). Light and fluorescence microscopy were performed using a Nikon Eclipse 80i microscope. Measurement of TUNEL-positive cells was performed in a blinded manner on eight randomly generated visual fields at magnification of 200×. Double labeling fluorescence techniques were used to evaluate TUNEL-positive cells in relation with occludin or ZO1 staining in the lung tissue sections. Briefly, paraffin-embedded sections were deparaffinized in xylene and rehydrated in 100, 95, and 70% ethanol. Next, the sections were heated at 95°C for 20 min in antigen retrieval buffer with sodium citrate (0.29g citrate + 0.1% Triton in 100 ml ddH₂O). Next, the sections were incubated for 30 min with proteinase K at 37°C and permeabilized with 0.3% Triton X-100/PBS (PBST). The TUNEL method was performed first (incubation for 1 h with TUNEL reaction mixture at 37°C in the dark), followed by washes with PBS. The tissue sections were blocked with protein block serum-free solution (DAKO) for 30 min in the dark. For occludin or ZO1 immunofluorescence staining, the sections were incubated for 1 h with a mouse monoclonal anti-occludin antibody (Clone OC-3F10) (1:100) or a rabbit polyclonal anti-ZO1 antibody (1:50) in PBS overnight at 4°C in a moist chamber in the dark. After being washed in PBS, the sections were incubated for 1 h in PBS containing Alexa Fluor 546-conjugated goat anti-mouse or Alexa Fluor 546-conjugated goat anti-rabbit (1:250), respectively, at room temperature in the dark. After washes in PBS, the sections were stained with DAPI (300 nM) and incubated for 5 min in the dark. The sections were then mounted with a fluorescent mounting medium and analyzed by light and fluorescence microscopy by acquiring sets of serial optical sections in both the fluorescence and the differential interference contrast mode.

Measurements in lung homogenates

For caspase-3 activity measurements, 20 mg of lung tissue were homogenized in lysis buffer provided by the Caspase-3/CPP32 Fluorometric Assay Kit (Biovision), according to manufacturer’s instructions. The fluorescence signal was measured using a fluorescence plate reader. For occludin and ZO1 protein measurements in mouse lungs, the membrane and cytosolic proteins from 20 mg of mouse lung tissue were separated and isolated using the Mem-PER™ Plus membrane protein extraction kit (Thermo Scientific). Protease and phosphatase inhibitors (Roche) were added to the permeabilization and solubilization buffers. Briefly, lung tissue was washed in a microcentrifuge tube with cell wash solution. The lung tissue was cut into small pieces and homogenized in sample tubes filled with ceramic beads and permeabilization buffer, using a MagNA lyser (Roche Life Science). To separate cytosolic and membrane fractions, the lung homogenates were incubated in permeabilization buffer for 10 minutes on ice and then centrifuged at 16,000 × g for 15 minutes at 4°C. The supernatant containing cytosolic proteins was separated from the pellet. To obtain the membrane proteins, the pellet was re-suspended in solubilization buffer and incubated for 30 minutes at 4°C, and then centrifuged at 16,000 × g for 15 minutes at 4°C. Aliquots of cytosolic and membrane protein fractions were stored at −80°C until used. Total protein concentrations of the membrane and cytosolic fractions were measured by using by the bicinchoninic acid method (Pierce™ BCA Protein assay, Rockford, IL). Occludin and ZO1 were measured in lung lysates by ELISA kits from Cloud Clone Corporation and Mybiosource, respectively. The sensitivities of these immunoassays were 0.132 ng/ml for occludin and 0.1 ng/ml for ZO1. The fluorescence signal was measured using a fluorescence plate reader.

Cell culture

Human pulmonary alveolar epithelial cells (HPAEpiC) isolated by ScienCell Research Laboratories from human lung tissue were provided by Innoprot. HPAEpiC are comprised of alveolar type I and type II epithelial cells, but type I cells are predominant, as the number of type II cells decreases with successive passages. HPAEpiC were grown in alveolar epithelial cell medium, which was buffered with HEPES and bicarbonate and supplemented with 2% heat-inactivated fetal bovine serum, 1% epithelial cell growth supplement (EpiCGS) and 1% penicillin/streptomycin solution (ScienCell Research Laboratories) at 37°C and 5% CO2. For the bioassay experiments and measurement of albumin permeability, the HPAEpiC cells were seeded in transparent collagen-coated transwell inserts with a membrane pore size of 0.4 μm and surface area of 0.33 or 1.12 cm² (Corning, Lowell, MA) at a density of 1×10⁴ cells/well or 2×10⁴ cells/well, respectively. The culture medium was added to the upper and lower compartment of each transwell, and the cells were allowed to reach 100% confluence, which was confirmed by phase contrast light microscopy. Next, medium containing recombinant human soluble FasL at concentrations of 0, 33.3, 100, 300 or 600 ng/ml (as specified in each experiment) or PBS was added to the upper and lower compartments. As indicated in each experiment, the cells were pre-incubated for 1.5 h with the caspase-3 inhibitor zDEVD.fmk (10 μM), the negative control of the caspase-inhibitors zFA.fmk (10 μM), the tyrosine kinase inhibitor genistein (30 μM) or vehicle (DMSO). After incubation for 2 h with FasL, the culture medium was removed from the upper and lower transwell compartments, spun at 200 g, and stored in individual aliquots at −20°C for cytokine measurements.

Measurement of FITC-albumin permeability in cultured alveolar epithelial cell monolayers

After 2 h of incubation with human recombinant FasL (with or without preincubation with zDEVD.fmk, zFA.fmk, or genistein), the permeability to FITC-albumin of the HPAEpiC cell monolayers was determined by the addition of medium supplemented with 0.05% FITC-albumin in the lower compartments. The fluorescence intensity was measured in the medium collected from the upper and the lower transwell compartments, using a fluorescence plate reader. The permeability ratio to albumin was expressed as a percentage of top FITC-albumin fluorescence relative to bottom FITC-albumin fluorescence, which reflects the passage of FITC-albumin into the upper compartment in each transwell.

Cell viability and caspase 3 activity in alveolar epithelial cell monolayers

Cell viability was assessed using the PrestoBlue assay (Invitrogen), which incorporates an oxidation/reduction indicator that fluoresces red in the presence of active cellular metabolism. Data are shown as the percentage of cell death, which was calculated as followed: cell death (%) = 100 × ([live cell fluorescence – experimental fluorescence]/live cell fluorescence). Untreated live cell fluorescence corresponds to the fluorescence of cells in medium only. Finally, the cells were lysed, and caspase-3 activity was measured in each well using a Caspase-3 Fluorometric Assay Kit (Biovision) and a fluorescence plate reader.

Protein and tyrosine kinase activity measurements in alveolar epithelial cell monolayers

IL-8 and IL-6 in the culture medium of HPAEpiC monolayers incubated in different conditions, as indicated in each experiment, were measured by ELISA (R&D system) according to manufacturer’s instructions. The sensitivity of the ELISA kit for IL-8 was 7.5 pg/ml and for IL-6 was 9.38 pg/ml. Tyrosine kinase activity was measured in cell lysates by using a universal tyrosine kinase assay kit from Takara-Clontech according to manufacturer’s instructions. In additional cultured cells, the occludin and ZO1 protein levels of the HPAEpiC monolayers, were measured in cell lysates by ELISA kits from Cloud-Clone Corp., following the manufacturer’s instructions. The sensitivity of the ELISA kits for Occludin was 0.063 ng/ml and for ZO1 was 0.059 ng/ml.

Immunocytochemistry and TUNEL technique

After treatment with human FasL or medium, the HPAEpiC cells seeded in transwell membranes were washed with pre-warmed PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The transwell membrane was gently cut and placed over a paraffin film with cells facing up. After 3 washes with PBS, the cells were permeabilized for 10 min with PBS containing 0.3% triton on ice. After washes with PBS, the cell monolayers were blocked with protein block serum-free (DAKO) for 30 min at room temperature. The cells were then incubated with a mouse monoclonal anti-occludin (clone OC-3F10) or a rabbit polyclonal anti-ZO1 antibody (1:200) overnight at 4°C in a moist atmosphere. Subsequently, the cell monolayers were incubated for 1.5 h with Alexa Fluor 546-conjugated goat anti-mouse or Alexa Fluor 546-conjugated goat anti-rabbit secondary antibody (1:250) at room temperature in the dark, stained with DAPI (300nM), and mounted with fluorescent mounting medium and analyzed by fluorescence microscopy. For the experiments combining the TUNEL method (to label DNA fragments) and the immunocytochemistry (to label occludin or ZO1 proteins), the cells were permeabilized as explained above, washed twice in PBS and incubated with TUNEL reaction mixture for 60 min at 37°C. After 3 washes in PBS, the same immunocytochemistry protocol was performed in the cells as explained above. The labels were visualized by fluorescence microscopy using a green wavelength (TUNEL), a red wavelength (occludin, ZO1) and a blue wavelength (DAPI).

Statistical analysis

Quantitative variables were reported as mean (95%CI) unless otherwise indicated. Differences between three or more groups were analyzed using one-way ANOVA followed by the Bonferroni’s post-hoc tests for variables with normal distribution, or the Kruskal-Wallis test followed by the Dunn’s test for those without a normal distribution. Differences between two groups were analyzed using the two-tailed unpaired Student’s t test for variables with a normal distribution, or the Mann-Whitney test for variables without a normal distribution. A logarithmic transformation (log10) was used to reduce the heterogeneity of variances when these were significantly different. The sample size was calculated in initial experiments that evaluated the changes in the concentration of occludin and ZO1 in the lungs of wild-type mice treated with FasL vs. PBS. We detected a 50% fall of the concentration of occludin and ZO1 in FasL-treated mice compared with control mice with a maximal SD of 1.8. Considering a power (1-β) of 90% and a significance level (α) of 5%, the calculated number of mice needed was from 8 to 10 per group. A P value less than 0.05 was considered statistically significant. The statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc.).

RESULTS

Human FasL increased pulmonary protein permeability, neutrophil recruitment and lung weight in mice

Intratracheal instillation of recombinant human soluble FasL (rh-sFasL) in wild-type mice resulted in increases of the concentration of IgM and of the number of polymorphonuclear (PMN) leukocytes, and in a decrease of the number of macrophages in BAL fluid collected 16 h after the instillation (Figure 1.A–C). These changes in wild-type mice were further accompanied by an increase of the lung weight-to-total body weight ratio (Figure 1.D) compared with control mice treated with PBS. In contrast, rh-sFasL instillation did not induce changes in Fas-deficient lpr mice. Control wild-type and lpr mice instilled with PBS had similar IgM concentration and number of PMN and macrophages in the BAL fluid at baseline (Figure 1.A–C).

Fig. 1. Effects of FasL on pulmonary protein permeability (A), cellular inflammatory responses (B, C) and left lung weight (D) in wild-type and Fas-deficient lpr mice in vivo.

Mice were treated with intratracheal instillation of recombinant human soluble FasL (rh-sFasL, 25 ng/g body wt), or PBS (as control), and then studied 16 h later. The graphs show the effect of rh-sFasL on (A) the concentration of IgM (a plasma protein of large size, 900 kDa), (B) the number of polymorphonuclear (PMN) leukocytes, and (C) the number of macrophages in bronchoalveolar lavage (BAL) fluid, and on (D) the left lung wt-to-body wt ratio. In the graphs, each dot represents an individual mouse (n=10 per group). Horizontal bars represent means. *P < 0.05 vs WT-PBS group.

Human soluble FasL altered the expression of occludin and ZO1 in mouse lungs in vivo

To determine whether the disruption of the tight junction proteins occludin and ZO1 was a potential mechanism responsible for the increase in protein permeability and lung edema formation induced by FasL, we first measured the concentrations of occludin and ZO1 in the membrane and cytosolic fractions of lung tissue extracts collected 16 h after the intratracheal instillation of rh-sFasL or PBS in wild-type and lpr mice. As expected, the membrane fraction was enriched in occludin and ZO1 proteins compared with the cytosolic fraction of the lung tissue extracts in all groups of mice (Figure 2.A, B and Figure 3.A, B). For both fractions, the concentrations of occludin and ZO1 were similar in wild-type and lpr mice treated with PBS (Figure 2.A, B and Figure 3.A, B), despite a non-significant trend for a reduced expression of occludin in the membrane fraction in lpr mice (Figure 2. A). Whereas the instillation of rh-sFasL in wild-type mice decreased the protein concentration of occludin in the membrane fraction and of ZO1 in both the membrane and cytosolic fractions, the instillation of rh-sFasL in lpr mice caused no changes in the protein levels of occludin or ZO1 in any of the fractions (Figure 2.A, B and Figure 3.A, B).

Fig. 2. Human soluble FasL alters the expression of occludin in the lungs of wild-type, but not in the lungs of lpr Fas-deficient mice.

Mice were treated with intratracheal instillation of recombinant human soluble FasL (rh-sFasL, 25 ng/g body wt), and then studied 16 h later. As control, wild-type (WT) and lpr Fas-deficient (lpr) mice were treated with PBS via intratracheal instillation. We measured the concentration of occludin in the membrane and cytosolic fractions in mouse lungs by ELISA (A and B, respectively). The merged images of fluorescence microscopy and light microscopy with differential interference contrast (DIC) show occludin protein signal (red) and cell nuclei (DAPI staining, blue) over the structure of the alveolar walls. Original image magnification X200. (C). Representative images at larger magnification (x400) show the decreased expression of occludin protein (red signal) in the lung of WT mice treated with rh-sFasL compared with mice treated with PBS (D). In the graphs, each dot represents an individual mouse (n=10 per group). n of 5 mice per group for the immunofluorescence analyses. Horizontal bars represent means. *P < 0.05 vs WT-PBS group

Fig. 3. Human soluble FasL alters the expression of ZO1 in the lungs of wild-type, but not in the lungs of lpr Fas-deficient mice.

Wild-type (WT) and lpr Fas-deficient (lpr) mice were treated with intratracheal instillation of recombinant human soluble FasL (rh-sFasL, 25 ng/g body wt), and then studied 16 h later. As control, WT and lpr mice were intratracheally instilled with PBS. We measured the concentration of ZO1 in the membrane and cytosolic fractions in mouse lungs by ELISA (A and B, respectively). The merged images of fluorescence microscopy and light microscopy with differential interference contrast (DIC) show occludin protein signal (red) and cell nuclei (DAPI staining, blue) over the structure of the alveolar walls. Original image magnification X200 (C). Representative images at larger magnification (x400) show the decreased expression of ZO1 protein (red signal) in the lung of WT mice treated with rh-sFasL compared with mice treated with PBS (D). In the graphs, each dot represents an individual mouse (n=10 per group). n of 5 mice per group for the immunofluorescence analyses. Horizontal bars represent means. *P < 0.05 vs WT-PBS group

Immunofluorescence techniques in lung tissue sections showed that occludin and ZO1 were homogenously distributed in the cells of the alveolar walls in wild-type and lpr mice treated with PBS as well as in lpr mice treated with rh-sFasL. In contrast, the lungs of wild-type mice treated with FasL showed areas with loss of occludin and ZO1 expressions in the alveolar walls (Figure 2.C, D and Figure 3.C, D).

Altogether, these data showed that intra-tracheal instillation of rh-sFasL caused changes in occludin and ZO1 proteins in the lung of wild-type mice, but not in Fas-deficient lpr mice, suggesting that human soluble FasL alters the expression and normal distribution of these TJ proteins through activation of Fas receptor in mouse lungs in vivo.

FasL-induced apoptosis is locally associated with altered expression of occludin and ZO1 in mouse lungs in vivo

Compared with PBS treatment, instillation of rh-sFasL significantly elevated the number of cells with nuclei containing DNA strand breaks (TUNEL-positive signal) and caspase-3 activity in the lungs of wild-type mice (Figure 4.A, B, respectively). Importantly, the merge of immunofluorescence images from FasL-treated wild-type mice showed that occludin and ZO1 protein signals were diminished in the alveolar walls only in areas with TUNEL positive cells (Figure 4.C). In contrast, no increase of caspase-3 activity or TUNEL positive cells were observed in lpr mice. These results suggest an association between FasL induced-apoptosis and the disruption of tight junction proteins in mouse lungs in vivo.

Fig. 4. Fas-induced apoptosis is locally associated with altered expression of occludin and ZO1 in mouse lungs in vivo.

Wild-type (WT) and Fas deficient (lpr) mice were treated with one intratracheal instillation of recombinant human soluble FasL (rh-sFasL, 25 ng/g body wt), or PBS (as control). At 16 h post-instillation, we measured (A) the number of nuclei containing DNA strand breaks (TUNEL-positive signal) in lung tissue sections, and (B) caspase-3 activity in the lung homogenates. Merged images of representative lung tissue sections from PBS or FasL-treated WT mice (C), showed that occludin and ZO1 protein signals (red) were diminished in the alveolar walls only in areas with TUNEL positive cells (green). Original image magnification X400. n of 5 mice per group for the immunofluorescence analyses. In the graphs, each dot represents an individual mouse (n= 10 mice per group). Horizontal bars represent means. *P < 0.05 vs WT-PBS group

Human FasL increases alveolar epithelial cell permeability prior to cell death.

To determine whether cell death by apoptosis was a requirement for the increase of alveolar permeability induced by FasL or there were other apoptosis-related events contributing to this alteration, we measured the protein permeability and cell death in HPAEpiC monolayers after 2 h of incubation with increasing concentrations (from 33 to 600 ng/mL) of a cytotoxic long form of human recombinant sFasL (Figure 5.A and B). Cells incubated with medium only or with a short form of human recombinant soluble FasL (300 ng/mL), which is not cytotoxic for epithelial cells (22), were used as controls. HPAEpiC expressed aquaporin-5 (AQ5), a water channel protein specifically expressed in alveolar type I cells in the lung, and showed the characteristic staining pattern of actin (Supplementary Figure 1). Incubation of the cell monolayers with 33, 100, 300 and 600 ng/mL of the long form of FasL resulted in dose-dependent increases of FITC-albumin permeability [28.4% (26.3–30.5) (p = 0.3703), 39.1% (36.5–41.1) (p = 0.0120), 49.9% (47.4–52.7) (p = 0.0001) and 71.5% (64.4–80.5) (p < 0.0001), respectively], compared with medium only [23.8% (23.1–24.6)] (Figure 5.A). The early alteration of transepithelial permeability by FasL was also observed when using the Transepithelial Electrical Resistance method (see Supplementary Figure 2). Importantly, the percentage of cell death at this 2-hour time point was only increased by the 300 ng/ml and 600 ng/ml doses of the long form of human sFasL [% cell death: 8.0% (5.5–10.5) (p = 0.022) and 25.2% (22.5–27.9) (p = 0.0025), respectively], compared with non-treated cells (Figure 5.B). Although all doses of sFasL resulted in apoptosis with long (> 6 h) incubation times (see Supplementary Figure 3), the 100 ng/ml dose of long sFasL did not cause detectable cell death despite enhancing protein permeability at the 2 h time point. Exposure to the non-cytotoxic short form of rh-sFasL (300 ng/mL) did not induce cell death nor changed the protein permeability of the alveolar epithelial cell monolayers (Figure 5.A, B). These data suggested that Fas signaling contributes to the loss of the alveolar epithelial barrier function by mechanisms preceding the ultimate death of the cells.

Fig. 5. Human soluble FasL increases alveolar epithelial cell permeability prior to cell death in vitro.

The permeability to FITC-albumin (A) and the percentage of cell death (B) were measured in human primary alveolar epithelial cell (HAEpiC) monolayers after 2 h of incubation with increasing concentrations of a cytotoxic long form of human recombinant sFasL from 33 to 600 ng/mL. Cells with medium only (MO) or with a non-cytotoxic short form of human recombinant soluble FasL (300 ng/mL) were used as controls. Results from 3 separate experiments performed in duplicate. Each dot of the graphs represents a single data point. Horizontal bars represent means. *P < 0.05 vs medium only (MO).

The increase in protein permeability induced by soluble FasL is dependent on caspase-3 but not on tyrosine-kinase signaling-dependent cytokine production.

To determine the signaling pathways by which human soluble FasL altered the lung epithelial barrier function, we performed experiments in HPAEpiC monolayers using the caspase-3 inhibitor zDEVD.fmk and the tyrosine-kinase specific inhibitor genistein.

First, we determined the minimal dose of zDEVD.fmk that blocked caspase-3 activity in HPAEpiCs by pre-incubating them for 1.5 h with different doses (from 1 to 100 μM) of zDEVD.fmk, its negative control peptide zFA.fmk, or vehicle (DMSO). Then, the cells were exposed for 2 h to medium only or to the two doses of rh-sFasL that increased protein permeability associated with no cell death (100 ng/ml) or only with minimal cell death (300 ng/ml). Compared with medium only, both doses of FasL increased the activity of caspase-3 in a dose-dependent manner in cells pre-incubated with vehicle [medium only: 30.9 (26.0–35.8) arb. units vs. FasL-100 ng/ml: 85.7 (56.5–114.9) arb. units (p = 0.0002) vs. FasL-300 ng/ml: 273.6 (242.1–305.1) arb. units (p < 0.0001)] or with zFA.fmk (Figure 6.A). In contrast, pre-incubation with zDEVD.fmk at the dose of 10 μM totally prevented the increase in caspase-3 activity caused by both doses of FasL (Figure 6.A). Remarkably, pre-incubation with zDEVD.fmk, but not with vehicle or zFA.fmk, also abrogated the increase in FITC-albumin permeability caused by both doses of FasL (Figure 6.C). Whereas the dose of 100 ng/ml of FasL did not cause cell death in any of the groups as measured by PrestoBlue™ reagent, the small percentage of cell death caused by the 300 ng/ml dose of FasL was also abolished in cells pre-incubated with zDEVD.fmk (Figure 6.B). Pre-incubation with vehicle, zFA.fmk or zDEVD.fmk in cells not exposed to FasL did not alter caspase-3 activity, cell viability or protein permeability (Figure 6.A–C). These data showed that FasL increased protein permeability of human alveolar epithelial cell monolayers via a caspase-3 dependent pathway before cell death occurred.

Fig.6. Human sFasL increases protein permeability in human alveolar epithelial cell monolayers by mechanisms dependent on Caspase-3, but not on tyrosine-kinase signaling-dependent cytokine production.

HPAEpiC monolayers were pre-incubated with caspase-3 inhibitor zDEVD.fmk (or zFA.fmk or vehicle as controls) and exposed for 2 h to rh-sFasL (100 ng/mL or 300 ng/mL) or medium only (MO) (A-C). After treatment, we measured (A) caspase 3-activity, (B) percentage of cell death and (C) FITC-albumin permeability in these cell monolayers. HPAEpiC monolayers were also pre-incubated with the tyrosine-kinase specific inhibitor genistein (or vehicle as control) (D-H) and exposed for 2 h to rh-sFasL (100 ng/mL or 300 ng/mL) or medium only (MO). After treatment, we measured IL-8 concentration in cell supernatant by ELISA (D), percentage of cell death by PrestoBlue (E), permeability to FITC-albumin (F), IL-6 concentration in cell supernatant by ELISA (G), and tyrosine kinase activity in the cell monolayers (H). Cell monolayers treated with medium only were used to determine 100% survival. Results from 4 separate experiments performed in duplicate. Each dot of the graphs represents a single data point. Horizontal bars represent means. *P<0.05 vs all conditions with MO; # P< 0.05.

Because it is known that Fas activation in lung epithelial cells increases cytokine production simultaneously to the induction of apoptosis (24), we investigated if cytokine production was involved in the sFasL-induced alteration of the lung epithelial barrier function. First, we confirmed that the production of IL-8 by HPAEpiC monolayers was increased as soon as 2 h after exposure to rh-sFasL at the doses of 100 and 300 ng/mL compared with medium only [FasL 100ng/ml: 9.28 pg/ml (8.54–10.02) (p < 0.0001), FasL 300 ng/ml: 10.58 pg/ml (9.58–11.58) (p < 0.0001) vs medium only: 5.28 pg/ml (4.71–5.84)]. In the following experiments, HPAEpiC monolayers were pre-incubated with different concentrations (from 10 to 100 μM) of genistein or vehicle for 1.5 h before exposure to rh-sFasL. The minimal dose of genistein tested that effectively prevented the increase in IL-8 expression induced by rh-sFasL without causing cell death was 30 μM (Supplementary Figure 4). In addition, pre-incubation with this dose of genistein (30 μM) significantly diminished tyrosine kinase activity in HPAEpiC treated for 2 h with rh-sFasL (100 or 300 ng/ml) or medium only (Figure 6.H). This was associated with a significantly decrease in the expression of IL8 and IL6 induced by FasL in these cells (Figure 6.D, G). Despite blocking cytokine production and tyrosine kinase activity, pre-incubation with genistein did not prevent or attenuate the increased protein permeability induced by the two doses of rh-sFasL (100 or 300 ng/ml) in human alveolar cell monolayers (Figure 6. F). Pre-incubation with 30 μM genistein did not cause nor influence sFasL-induced cell death either (Figure 6.E). These results suggested that tyrosine kinase activity and cytokine production were not required for the early increase in protein permeability induced by sFasL in alveolar epithelial cell monolayers.

Human sFasL alters the levels and distribution of occludin and ZO1 proteins by caspase-3-dependent, tyrosine kynase-independent mechanisms.

To determine whether the increase of protein permeability induced by sFasL in human primary alveolar epithelial cell (HAEpiC) monolayers was associated to alterations of tight junction proteins, we assessed protein permeability and the concentration and distribution of occludin and ZO1 in HPAEpiC monolayers 2 h after exposure to rh-sFasL (100 ng/ml) or medium only. Compared to control cells, the exposure to rh-sFasL decreased the concentrations of occludin and ZO1 in cell protein extracts, as measured by ELISA (Figure 7.A, B). Immunocytochemical evaluation of these human alveolar epithelial cells, predominantly type I cells, showed occludin signal mainly distributed along the cytoplasmic extensions of the cells but not exclusive at the membrane sites of cell-cell contacts as occurs in cuboidal epithelial cells. ZO1 signal was mainly found at the nuclei, and distributed along the cytoplasmic extensions and at sites of cell-cell contact. Exposure to rh-sFasL resulted in a global decrease of occludin and ZO1 fluorescence that was particularly profound in the cytoplasmic extensions, while some signal remained close to the cell nuclei (Figure 7.C, D). Pre-incubation with zDEVD.fmk effectively prevented the sFasL-induced decrease of occludin and ZO1 proteins evaluated both by ELISA (Figure 7.A, B) and by immunocytochemistry (Figure 7.C). In contrast, pre-incubation with genistein or its vehicle (DMSO) or with zFA.fmk, had no protective effects (Figure 7. A–C). Of note, pre-incubation with vehicle, genistein, zFA.fmk or zDEVD.fmk alone did not alter the expression of these TJ proteins (data not shown).

Fig. 7. Human sFasL alters the levels and distribution of occludin and ZO1 proteins in human lung alveolar epithelial cells in vitro by mechanisms dependent on caspase-3, but independent of tyrosine kinase-mediated cytokine production.

After 2 h exposure of rh-sFasL (+FasL) at the dose of 100 ng/mL or medium only (-FasL), the concentrations of occludin and ZO1 proteins were measured by ELISA in HAECpiC monolayers pre-incubated with the caspase-3 inhibitor zDEVD.fmk, its inactive analog (zFA.fmk), or the tyrosine kinase inhibitor genistein or vehicle (DMSO). Compared to control cells, rh-sFasL decreased the concentrations of occludin and ZO1 in cell protein extracts (A and B, respectively). Representative immunofluorescence images (original image magnification X400) (C) show the expression of occludin and ZO1 proteins (red signals) localized along the cytoplasmic membrane of control cells in HAECpiC monolayers (−rhFasL/+vehicle). Exposure to rh-sFasL resulted in a global decrease of occludin and ZO1 fluorescence signals that was particularly profound in the cytoplasmic extensions, while some signal remained close to the cell nuclei (DAPI staining, blue signal). Only pre-incubation with the caspase-3 inhibitor zDEVD.fmk prevented the sFasL-induced decrease of occludin and ZO1 proteins evaluated both by ELISA (A and B) and by immunocytochemistry (C). Representative immunofluorescence images at larger magnification (x1000) show the decreased expression of occludin and ZO1 proteins (red signals) in HAECpiC monolayers treated with rh-sFasL (+rh-sFasL/+vehicle) compared with control cells (−rh-sFasL/+vehicle) (D) Results from 4 separate experiments performed in duplicate. Each dot of the graphs represents a single data point. Horizontal bars represent means. *P<0.05 vs their corresponding-rh-sFasL conditions.

Finally, to determine whether the loss of occludin and ZO1 proteins induced by sFasL was an early event in the process of apoptosis, we incubated HPAEpiC monolayers with rh-sFasL (100 ng/mL) for 1, 2, 3 or 5 h. By immunocytochemistry, we observed that the fluorescence signal of both occludin and ZO1 proteins began to fall at least 2 h before DNA fragmentation occurred in the nuclei of those cells, which is one of the last events in the apoptotic process (Figure 8. A, B).

Fig. 8. FasL-mediated changes in the levels and distribution of occludin and ZO1 proteins occur before nuclear DNA fragmentation in human lung alveolar epithelial cells in vitro.

Representative fluorescence images of HAECpiC monolayers exposed to rh-sFasL (100 ng/mL) or medium only (MO) for 1, 2, 3 or 5 h. The cells were labeled with the TUNEL method for the detection of DNA fragmentation (green signal), followed by labelling with an antibody to occludin or ZO1 proteins (red signal). The merged image shows a decrease of occludin and ZO1 fluorescence signals after exposure to FasL in a time-dependent manner. Despite this loss of occludin and ZO1 fluorescence signal, no TUNEL positive signal was observed during the first 3 h of FasL exposure. However, TUNEL positive cells were observed at 5 h-exposure of FasL. Original image magnification X400.

DISCUSSION

The main goal of the present study was to determine whether Fas activation alters the alveolar epithelial function by mechanisms involving disruption of TJs in the lung. Our results demonstrated that activation of the Fas/FasL system altered TJ proteins and increased protein permeability of the alveolar-capillary membrane in mouse lungs in vivo. In alveolar epithelial cell monolayers, FasL caused a rapid alteration of TJ proteins associated with a marked increase in protein permeability prior to cell death by mechanisms dependent on caspase-3 activity. These data indicate that the Fas/FasL system increases pulmonary protein permeability by a direct effect on the alveolar epithelium that involves the alteration of its TJ proteins and permeability properties.

Our previous studies suggested that Fas-mediated apoptosis in alveolar epithelial cells impairs alveolar fluid clearance and increases pulmonary permeability by caspase-dependent mechanisms, leading to lung edema formation in mouse lungs in vivo (23). This occurred in spite of a relatively small number of apoptotic cells observed in most animal models of acute lung injury involving the Fas/FasL system, such as those induced by endotoxin, bleomycin or mechanical ventilation (29). Although this situation could be explained by a rapid clearance of apoptotic cells, it is conceivable that the activation of apoptotic pathways also causes cellular changes that may contribute to lung edema by mechanisms that do not depend on the ultimate death of epithelial cells in the lung. Reduced expression of TJ proteins has been identified in these animal models of acute lung injury together with an important dysfunction of the alveolar-capillary membrane (9, 10, 30, 31). Those observations led us to explore whether the disruption of TJ proteins can be a mechanism by which Fas/FasL leads to lung edema formation in acute lung injury, besides the loss of cells mediated by apoptosis.

In the present study, activation of the Fas/FasL system decreased the expression of two TJ proteins, occludin and ZO1, in the alveolar walls of mouse lungs in vivo, which was associated with a significant increase in protein permeability and lung edema formation, whereas mice lacking functional Fas receptor (lpr mice) were protected. Changes in the distribution of these TJ proteins occurred only in wild-type mice after FasL instillation with a tendency to form aggregates in the alveolar walls. Whereas the loss of ZO1 protein results in a significant increase in epithelial protein permeability in most studies, a decreased expression of occludin is not always required to alter the barrier function in several non-pulmonary epitheliums (32). In line with this, the present study showed that the lower levels of occludin observed in lpr mice compared with wild-type mice was not associated with alveolar barrier dysfunction at baseline, which may be explained by their similar levels of ZO1 proteins.

In human alveolar epithelial cells, a 2 h-exposure to human sFasL increased protein permeability of the alveolar epithelial monolayers together with a decrease in occludin and ZO1 protein expression. These events occurred along with a prompt activation of the pro-apoptotic casaspe-3 enzyme, but the cells were still alive and no cells were detached from the matrix after 2 h-treatment with sFasL. In addition, the morphology of these cells and the cell-cell contacts were similar to the untreated-cells, except for a slight nuclear condensation. This is similar to other studies in which induction of apoptosis by other stimuli such as UV-irradiation, staurosporine or anoxia resulted in reduced expression of occludin and/or ZO1 proteins in non-pulmonary epithelial or endothelial cell lines in vitro as early as 6 h, 3 h and 30 min, respectively (13, 33, 34). These observations also occurred while the cells acquired a more rounded shape, but they were still attached, and no cell death was detectable by the cell viability assay (Prestoblue). In addition, we observed in a time-course experiment with HPAEpiC monolayers that exposure to FasL diminished that expression of occludin and ZO1 at least 2 h before nuclear DNA fragmentation (TUNEL positive) occurred. Also, cells showing important reduction of occludin and ZO1 fluorescent signal but without DNA fragmentation (TUNEL negative) were detected. All these data indicated that disruption of TJ proteins was an early event of FasL-mediated apoptosis that occurred prior to nuclear DNA fragmentation. Importantly, this prompt disruption of TJ protein induced by FasL was capable of increasing protein permeability of the alveolar epithelium before complete cell death was established, and therefore while the cells were still alive. We cannot discard, however, that the ultimate death of cells can also contribute to a further increase in protein permeability of the alveolar epithelial cell monolayers caused by the activation of the Fas/FasL system.

In our study, blockage of caspase-3 by using zDEVD.fmk prevented the deleterious effect of human FasL on TJ protein expression and on protein permeability in human alveolar epithelial cell monolayers in vitro. This is in agreement with our previous studies in which broad blockage of caspases significantly attenuated the increase in protein permeability and decreased apoptosis induced by human sFasL in mouse lung (23). There is also evidence that TJ proteins, such as occludin, ZO1 and ZO2 are substrates of caspase-3 and other caspases after different pro-apoptotic stimuli, including Fas activation, in non-pulmonary apoptotic epithelial cells (13, 14, 35). Also, occludin has been found in the death-inducing signaling complex (DISC), where it colocalized with Fas, Fas-associated protein with death domain (FADD), active caspase-8 and caspase-3 (35, 36). This may explain the relationship found in our study between caspase-3 activation and disruption of TJ proteins after Fas/FasL-mediated apoptosis in alveolar epithelial cells.

Activation of Fas via instillation of FasL not only induces apoptosis in mouse lungs, but it also activates inflammatory responses with neutrophil recruitment to the alveolar spaces and increase in cytokine production in the lung (22). Instillation of FasL into the lung also reduces the number of macrophages in the bronchoalveolar lavage fluid, compared with PBS treated mice. This could be explained by a potential Fas-mediated activation of macrophages in the airspaces, because activated macrophages have been shown to adhere and interact with the epithelium, hindering their extraction by the bronchoalveolar lavage. Isolated mouse alveolar epithelium and human distal airway epithelial cells exposed to FasL or to the Fas activating Jo2 antibody undergo apoptosis through caspase-3 dependent pathways, along with an increase in KC/IL-8 release by mechanisms involving a rapid phosphorylation of several kinases, such as MAPK, ERK and JNK. This rapid kinase phosphorylation was detected as early as 30 min after Fas activation in vitro (24). Also, it has been shown that Fas receptor physically interacts with tyrosine kinases, leading to a rapid cytokine production in non-pulmonary epithelial cells upon Fas activation (37, 38). Interestingly, it has been shown that occludin also binds to a complex set of tyrosine kinases (39, 40). All these data point out a relationship between Fas receptor, tyrosine kinases, cytokine production and occludin protein. By using genistein, a tyrosine kinase inhibitor, we diminished the activity of tyrosine kinase and blocked the early expression of IL-8, but we could not prevent the loss of TJ proteins -occludin or ZO1- or the enhanced protein permeability caused by human sFasL in human alveolar epithelial cell monolayers. These observations suggest that the expression of cytokines mediated by tyrosine kinases does not play a major role in the initial disruption of TJ proteins and loss of barrier function of the alveolar epithelium caused by human FasL. Because tyrosine kinase activation may also be involved in Fas-mediated apoptosis, we cannot discard that tyrosine kinases contribute to Fas-mediated alteration of TJ and epithelial permeability in later time-points.

In the present study, we have used the irreversible inhibitor of caspase-3 zDEVD.fmk, but it can also have some inhibitory activity on other caspases, such as caspase-6, −7, −8 and −10, all of them involved in the apoptotic pathway. In contrast, it cannot inhibit any of the caspases involved in cytokine activation (caspase-1, −4, −5, and −13). Therefore, we can assume that the effects of zDEVD.fmk were derived from the specific blockage of the caspase-dependent apoptotic pathway. Genistein is a highly specific inhibitor of protein tyrosine kinase (PTK) by preventing tyrosine phosphorylation. To reduce unwanted potential effects of genistein on cell death, we used the minimal dose of genistein that significantly prevented the increase of IL8 induced by FasL without causing cell death (30 μM). Regarding the doses of sFasL used in our study, we believe that they do not differ from the concentration of sFasL found in the alveoli in patients with ARDS. Based on previous studies in these patients (Matute-Bello G et al. J Immunol. 1999 Aug 15;163(4):2217–25), the alveolar concentration of sFasL could range from 15 to 150 ng/ml, which agrees with the doses that we used.

Our study has some potential limitations. Firstly, we evaluated mouse lungs in vivo and human alveolar epithelial cells in vitro, so caution must be exercised when extrapolating our findings to human lungs. Secondly, it is known that the extracellular matrix influences the expression of tight-junction proteins in the epithelium. Although we used unmodified human primary alveolar epithelial cells, the response to FasL of the cells seated on collagen-coated membranes might differ from the real alveolar epithelium seated in the more complex basement membrane of human lungs. In addition, we did not assess other factors such as oxidative stress or mitochondrial damage that could also contribute to alveolar damage. Thirdly, we have focused on alveolar epithelial cells, but Fas activation might also influence the expression of tight-junction proteins in the endothelium. Finally, we cannot discard that apoptosis and inflammation also influence the expression and function of TJ proteins at time points later than those evaluated in our study.

In conclusion, activation of pro-apoptotic caspase-3-dependent pathways of the Fas/FasL system is an important mechanism responsible for the alteration of TJs - occludin and ZO1- in the alveolar-capillary membrane that contributes to lung edema formation. The disruption of TJs in the alveolar epithelium caused by Fas/FasL is accompanied by an increase in the permeability of the alveolar epithelium, long before the epithelial cells die by apoptosis. We suggest that Fas/FasL activation leads to lung edema by causing an important disruption of the alveolar epithelial TJs followed by cell death in the alveolar walls. These data also provide a basis for further studies designed to determine whether the inhibition of the Fas/FasL pathway may prevent lung edema formation and limit the progression of diffuse alveolar damage in ARDS, particularly in the earliest stage of the disease when denudation of the alveolar epithelium is minimal. Therapeutic strategies aimed to enhance proliferation and differentiation of the alveolar epithelial cells may be a better option at later stages when denudation of the alveolar epithelium is present.

What is the key question?

The key question of our study is whether the alteration of tight junction proteins in the alveolar epithelium is a mechanism by which the activation of the Fas/FasL system increases pulmonary permeability in acute lung injury, even in the absence of apoptotic cell death.

What is the bottom line?

The activation of the Fas/FasL system is capable of increasing protein permeability of the alveolar epithelium in vivo and in vitro by mechanisms involving caspase-3-dependent alterations of tight junction proteins (occludin and ZO1) prior to and/or independently of the development of cell death.

Why read on?

This is the first study that links Fas activation with the alterations of tight junction proteins in the pulmonary alveolar epithelium in acute lung injury, suggesting that targeting this pathway may be a suitable strategy to preserve the alveolar epithelial function and to reduce lung edema in acute lung injury.