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Am. J. Respir. Cell Mol. Biol., Volume 21, Number 3, September 1999 436-445

Induction of Interleukin-8 Secretion and Apoptosis in Bronchiolar Epithelial Cells by Fas Ligation

Naoki Hagimoto, Kazuyoshi Kuwano, Masayuki Kawasaki, Michihiro Yoshimi, Yumi Kaneko, Ritsuko Kunitake, Takashige Maeyama, Takuo Tanaka, and Nobuyuki Hara

Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, Fukuoka, Japan


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Epithelial cell injury is the common manifestation of lung injury. Contributing to such injury of epithelial cells is apoptosis. Although apoptosis is part of the normal process of epithelial renewal, in excess it is pathologic. We previously demonstrated the excessive apoptosis of lung epithelial cells and the upregulation of Fas and Fas ligand (FasL) in fibrosing lung diseases. We also showed that inhalation of anti-Fas antibody induced lung injury and fibrosis in mice. Interleukin (IL)-8 is one of the most important cytokines in the pathophysiology of acute lung injury and pulmonary fibrosis. In this study we investigated whether Fas ligation induces IL-8 secretion in addition to apoptosis in bronchiolar epithelial cells in vitro. Bronchiolar epithelial cells underwent apoptosis and also secreted IL-8 in response to tumor necrosis factor (TNF)-alpha or Fas ligation. New gene expression and protein synthesis were not necessary for Fas ligation- and TNF-alpha - mediated apoptosis, but were necessary for IL-8 secretion. We further found that Fas ligation induced activation of nuclear factor-kappa B. We conclude that the Fas/FasL pathway not only mediates apoptosis but also plays a proinflammatory role, and that stimulation of the Fas/FasL pathway in bronchiolar epithelial cells leads to IL-8 production, which may amplify the inflammatory cascade in lung injury and pulmonary fibrosis.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Fas antigen (Fas), a type I membrane receptor protein and a member of the tumor necrosis factor (TNF) receptor family (1), induces apoptosis after engaging with Fas ligand (FasL) (2). Fas is expressed in various cells and tissues, including the thymus, liver, skin, heart, and lung (3- 5). Other investigators have suggested that loss of epithelial cells through the Fas pathway might play an important role in tissue injury or organ dysfunction (3, 6). Damage to and loss of epithelial cells are also commonly seen in acute lung injury (ALI) and in chronic fibrosing alveolitis.

We previously showed that the expression of Fas was upregulated in bronchiolar and alveolar epithelial cells, and that FasL was expressed in infiltrating lymphocytes in lung tissues from patients with idiopathic pulmonary fibrosis (IPF) (7). In bleomycin-induced pulmonary fibrosis, Fas and FasL were upregulated in lung epithelial cells and infiltrating lymphocytes, respectively, and apoptosis of bronchiolar and alveolar epithelial cells was detected (8). Other recent studies have suggested a role of the Fas signaling pathway in apoptosis of alveolar epithelial cells in vitro (5, 9). Furthermore, inhalation of anti-Fas antibody (Jo2) induced lung injury and fibrosis in mice (10). Seino and coworkers suggested that FasL may also play a proinflammatory role, as observed in their study (11).

Interleukin (IL)-8 is a cytokine with potent chemotactic properties for neutrophils and T lymphocytes, and thus serves to amplify the inflammatory cascade (12, 13). IL-8 is produced by a wide variety of cell types including mononuclear cells, endothelial cells, and epithelial cells (14), and has been implicated as an important mediator of neutrophil infiltration in lung tissues from patients with IPF (15, 16) or ALI (17).

IL-8 is produced in response to TNF-alpha in primary cultured human airway epithelial cells and in a human lung epithelial cell line (21, 22). Recent studies have shown that several members of the TNF receptor (TNFR) family activate the transcription factor nuclear factor-kappa B (NF-kappa B) through a common adapter protein, and regulate NF-kappa B-dependent cytokine production (23).

In this study we investigated whether Fas ligation induces IL-8 production in addition to apoptosis in bronchiolar epithelial cells in vitro, comparing this with the effect of TNF-alpha with or without interferon (IFN)-gamma . We also investigated whether Fas ligation induces NF-kappa B activation in these cells. In addition to apoptosis, a proinflammatory role in tissue injury may be another function of the Fas- FasL pathway, and may be a mechanism by which Fas ligation induces lung injury and pulmonary fibrosis.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell Culture

Cryopreserved primary human bronchiolar epithelial cells (Cryo SAEC) were purchased from Clonetics Corp. (San Diego, CA) and were used between passages 2 and 5 in this study because the cells' growth, sensitivity to Fas ligation, and cytokine secretion were constant during these passages (data not shown). As previously described, cell- matrix and cell-cell interactions modulate apoptosis in vitro (26). In this study, Cryo SAEC cells were grown in 25-cm2 tissue culture flasks (Falcon, Franklin Lakes, NJ) in small-airway epithelial-cell growth medium (SAGM, Clonetics) supplemented with hydrocortisone (0.5 µg/ml), bovine pituitary extract (30 µg/ml), epidermal growth factor (0.5 ng/ml), epinephrine (500 ng/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ml), gentamicin (50 µg/ml), amphotericin B (50 ng/ml), and 5% bovine serum albumin. The cultures were incubated at 37°C in a humidified, 95% air/5% CO2 atmosphere. After trypsinization, the cells were subcultured into six-well culture plates (Falcon) or 25-cm2 tissue culture flasks at a seeding density of 1 × 105 cells/cm2. When the cells were 50 to 60% confluent, the medium was changed to SAGM without hydrocortisone and the cells were allowed to grow for an additional 24-48 h before addition of particles, until they reached 70-80% confluence.

Reagents and Antibodies

Human recombinant TNF-alpha and IFN-gamma were obtained from Dainippon Pharmaceutical Co. Ltd. (Tokyo, Japan) and from Shionogi Ltd. (Tokyo, Japan), respectively. A monoclonal antihuman Fas antibody for the induction of apoptosis (clone CH-11), a monoclonal antibody for neutralization of the Fas signal (clone ZB-4), and isotype-matched mouse IgM as a control for the CH-11 antibody were purchased from MBL (Nagoya, Japan). Fluorescein isothiocyanate (FITC)-labeled anti-Fas antibody (clone UB2) and phycoerythrin (PE)-labeled monoclonal antibody Apo2.7 (clone 2.7A6A3) for flow cytometry were purchased from Coulter, Inc. (Miami, FL). ZB-4 was used at a concentration of 10 µg/ml and given 2 h before TNF-alpha or CH-11. The metabolic inhibitors actinomycin D (Sigma Chemical Co., St. Louis, MO) and cycloheximide (Sigma) were added at concentrations of 1 µg/ml and 10 µg/ml, respectively, when CH-11 or TNF-alpha was administered.

Flow Cytometric Analysis of Fas

For analysis of Fas surface expression on bronchiolar epithelial cells, unstimulated cells and cells treated with TNF-alpha (4 ng/ml) for 24 h, IFN-gamma (40 ng/ml) for 6 h, or TNF-alpha with IFN-gamma pretreatment were removed from the plate with 5 mM ethylenediaminetetraacetic acid (EDTA), pelleted, and resuspended in a staining solution containing phosphate-buffered saline (PBS) with 1% fetal calf serum (FCS). Cells were labeled with 1 µg/ml of FITC-conjugated anti-Fas antibody (UB2) or control FITC-conjugated mouse IgG1 (MBL) for 45 min at 4°C. Ten thousand viable cells were analyzed on an EPICS XL flow cytometer (Coulter).

Measurement of IL-8 Secretion

Bronchiolar epithelial cells were allowed to attach in six-well flat-bottom culture plates at a density of 105 cells/well. TNF-alpha or CH-11 was added after 24 h. Supernatants were harvested at 6, 12, 24, and 48 h after TNF-alpha or CH-11 administration, and were assessed for IL-8 concentration. If preincubation with IFN-gamma was done, cells were exposed to IFN-gamma (40 ng/ml) for 6 h, washed three times, and kept in fresh medium overnight. TNF-alpha or CH-11 was added on the following day, and supernatants were harvested after 24 h. The concentration of IL-8 in the culture supernatants was measured with enzyme-linked immunosorbent assay (ELISA) kits (Amersham, Buckinghamshire, UK) according to the manufacturer's instructions.

Analysis of DNA Fragmentation

Genomic DNA was obtained by lysing cells (106 cells) in a mixture of 100 mM Tris-HCl (pH 8.0), 40 mM Na2EDTA, 10 mM NaCl, 1% sodium dodecylsulfate (SDS), and 500 ng/ml proteinase K (Amresco, Inc., Solon, OH). The samples were incubated at 50°C overnight, and were then subjected to phenol-CHCl3 (1:1) extraction. After ethanol precipitation, the DNA was dried. The pellet was resuspended in 30 µl of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. DNA was electrophoresed on a 1% agarose gel, stained briefly with ethidium bromide, and photographed under ultraviolet transillumination.

Quantification of Apoptosis through Flow Cytometry

Apo2.7 antibody reacts with a 38-kD mitochondrial membrane protein (7A6 antigen) that appears to be exposed on cells undergoing apoptosis. It has been suggested that the Apo2.7 protein is involved in the molecular cascade of apoptosis, and that its expression represents an early event in apoptosis rather than a final product of dead cells (27).

Cells were stained with the Apo2.7 antibody as described previously (27, 28), with slight modifications. In brief, a stock solution of 25 mg/ml digitonin (Sigma) was prepared by adding it to PBS and heating to 100°C until completely dissolved. Bronchiolar epithelial cells, treated with each agonist or antibody, were permeabilized in 100 µg/ml digitonin solution diluted from stock solution in PBS, and were then incubated in this solution for 20 min on ice. Cells were washed and stained with 2 µg/ml of either PE-labeled APO2.7 antibody or with control antibody for 15 min. Cells were washed with 1.0 ml of PBS containing 2.5% FCS, and were stored in the dark on ice until analyzed with a flow cytometer. Ten thousand events were collected and analyzed flow cytometrically. The relative number of Apo2.7-positive cells were determined by setting the analytical region to a more fluorescent population that exceeded the upper limit of fluorescence set with nonapoptotic specimens.

Electron Microscopy

The cells treated with CH-11 (500 ng/ml) for 48 h were harvested after centrifugation and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 18 h. The cells were postfixed for 1.5 h in 1% OsO4 dissolved in 0.1 M phosphate buffer (pH 7.4), and were dehydrated through a series of graded ethanol solutions and then embedded in Epon. Ultrathin sections were cut, stained with uranyl acetate and lead nitrate, and examined under a JEM-1200 EX transmission electron microscope (JEOL Co., Tokyo, Japan).

Nuclear Extract Preparation

Nearly confluent cells, which had been treated with 4 ng/ml of TNF-alpha , 100 ng/ml of CH-11, or 500 ng/ml of control IgM for 2 h, were washed with ice-cold PBS, harvested by scraping into 1 ml of PBS, and pelleted in a 1.5-ml microfuge tube at 6,000 rpm for 5 min. The pellet was washed twice in ice-cold PBS and then suspended in one packed-cell volume of lysis buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine-N'-2 ethanesulfonic acid [Hepes], pH 7.9; 10 mM KCl; 0.1 mM EDTA; 1.5 mM MgCl2; 0.25 volume% Nonidet-P40; 1 mM dithiothreitol [DTT]; and 0.1 mM phenylmethylsulfonyl fluoride [PMSF]). After a 5-min incubation on ice, the nuclear pellet was isolated by centrifugation. The nuclear pellet was resuspended in one packed-cell volume of extract buffer (20 mM Hepes, pH 7.9; 420 mM NaCl; 0.1 mM EDTA; 1.5 mM MgCl2; 25% [vol/vol] glycerol; 1 mM DTT; and 0.5 mM PMSF) and the nuclei were incubated on ice for 20 min. The nuclear debris was removed by centrifugation and the protein concentration of the nuclear extract was determined. The nuclear extracts were stored at -80°C until further use.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay (EMSA) was performed with the DIG Gel Shift Kit (Boehringer-Mannheim, Indianapolis, IN). In brief, nuclear protein-DNA binding studies were done for 15 min at room temperature in a 20-µl reaction volume containing 20 mM Hepes, pH 7.6; 30 mM KCl; 1 mM EDTA; 1 mM DTT; 10 mM (NH4)2SO4; 1 µg polydeoxyinosine-deoxycytosine; 0.1 µg poly-L-lysine; 1% Tween 20 (wt/vol); 1 fmol digoxigenin-labeled DNA probe, and 5 µg nuclear protein. A consensus double-stranded NF-kappa B oligonucleotide (5'-AGT TGA GGG GAC TTT CCCAGG C-3'; Promega Corp., Madison, WI), which was labeled at the 3' end with digoxigenin, was used as a DNA probe. A 6% nondenaturing polyacrylamide gel was used for electrophoretic separation. After blotting on a membrane, labeled oligonucleotide was subsequently detected with an enzyme immunoassay, using antidigoxigenin alkaline phosphatase, Fab fragments, and the chemiluminescent substrate, 3-(4-methoxyspiro[1,2-dioxetane-3,2' - (5' - chloro) tricyclo [3.3.1.1.3, 7] decan]-4-yl) phenylphosphate disodium salt (DSPD) (Boehringer-Mannheim). The specificity of the binding reaction was tested by using an excess of unlabeled consensus NF-kappa B oligonucleotide to compete with the labeled probe for binding to the nuclear protein. Supershift assays with a polyclonal p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were done to confirm the presence of NF-kappa B p65 as part of the complex.

Statistics

All statistical data are expressed as the mean ± SE from at least three independent experiments. One-way analysis of variance (ANOVA) was done with StatView version 4.5 (Abacus Concepts, Inc., Berkeley, CA).

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Fas Is Expressed in Primary Bronchiolar Epithelial Cells

Figure 1 shows the result of flow cytometry for Fas expression in bronchiolar epithelial cells. Fas was expressed constitutively in these cells (Figure 1a). Fas expression was markedly upregulated at 24 h after incubation with TNF-alpha following preincubation with IFN-gamma (Figure 1d), but only slightly upregulated after incubation with IFN-gamma (Figure 1b) or TNF-alpha (Figure 1c) alone. We also examined FasL expression, but did not observe it on these cells (data not shown).


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  		Figure 1.   Fas expression on the surface of bronchiolar epithelial cells as determined with flow cytometry. (a) Nonstimulated bronchiolar epithelial cells stained with control IgG1 (solid line) or anti-Fas antibody (shaded area). (b) Bronchiolar epithelial cells stimulated by IFN-gamma (40 ng/ml) for 6 h were stained with control IgG1 (solid line) or anti-Fas antibody (shaded area). (c) Bronchiolar epithelial cells stimulated by TNF-alpha (4 ng/ml) for 24 h were stained with control IgG1 (solid line) or anti-Fas antibody (shaded area). (d) Bronchiolar epithelial cells pretreated with IFN-gamma (40 ng/ml) for 6 h and stimulated by TNF-alpha (4 ng/ml) for 24 h were stained with control IgG1 (solid line) or anti-Fas antibody (shaded area). The data are representative results from three experiments.

Apoptosis in Response to TNF-alpha and Fas Ligation

Agarose gel analysis showed that DNA fragmentation was present in DNA samples extracted from cells treated with CH-11 (100 ng/ml) or TNF-alpha (4 ng/ml) for 24 h and 48 h (Figure 2). The fragmented DNA did not show the typical pattern of oligonucleosomal fragmentation (180 to 200 bp). However, it has been reported that apoptosis in some epithelial cells can result in large DNA fragments before the appearance of or in the absence of small fragments (30, 31).


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  		Figure 2.   DNA fragmentation analysis on agarose gel electrophoresis. Genomic DNA obtained from bronchiolar epithelial cells treated with CH-11 or TNF-alpha . Large DNA fragments were seen in DNA extracted from bronchiolar epithelial cells treated with CH-11 (100 ng/ml) or TNF-alpha (4 ng/ml) for 24 h and 48 h. The data are representative results from three experiments.

Bronchiolar epithelial cells were monitored for early cellular effects of apoptosis by staining with PE-labeled Apo2.7 antibody. The results of flow cytometric analysis showed that CH-11 treatment induced apoptosis in 23% (100 ng/ml for 24 h), 49% (100 ng/ml for 48 h), and 78% (500 ng/ml for 48 h) of bronchiolar epithelial cells (Figure 3). Treatment with TNF-alpha or CH-11 induced apoptosis in a dose-dependent manner (Figure 4a). Figure 4b shows the time course of apoptosis of bronchiolar epithelial cells in response to TNF-alpha or CH-11. The number of apoptotic cells induced by Fas ligation was greater than that induced by TNF-alpha within 12 h. Although the agarose gel analysis did not show DNA fragmentation in DNA samples extracted from cells treated with CH-11 at 6 h to 12 h, flow cytometric analysis with PE-labeled Apo2.7 antibody did reveal apoptotic cells within this period. These results indicate that Apo2.7 detection with flow cytometry is more sensitive than DNA fragmentation analysis on agarose gel for detection of the early stage of apoptosis.


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  		Figure 3.   Flow-cytometric detection of PE-labeled Apo2.7 in CH-11-induced bronchiolar epithelial cell apoptosis. Bronchiolar epithelial cells were permeabilized with 100 µg/ml digitonin prior to staining with PE-labeled Apo2.7. The cells were then treated with 500 ng/ml control IgM for 24 h (a), 100 ng/ml CH-11 for 24 h (b), 100 ng/ml CH-11 for 48 h (c), or 500 ng/ml CH-11 for 48 h (d). The data are representative results from three experiments.


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  		Figure 4.   Induction of apoptosis in bronchiolar epithelial cells by CH-11 or TNF-alpha . (a) Concentration dependency. Cells were cultured with the indicated concentration of CH-11 or TNF-alpha for 24 h, and the proportion of cells undergoing apoptosis was quantified by flow cytometry done with PE-labeled Apo2.7. (b) Time course. Cells were cultured with CH-11 (100 ng/ml) or TNF-alpha (4 ng/ml) for indicated intervals with bars showing the percentage of cells undergoing apoptosis. Results are shown as mean ± SE of triplicate measurements. (* P < 0.01 versus control IgM).

Electron microscopy demonstrated that bronchiolar epithelial cells treated with CH-11 (500 ng/ml) for 48 h contained single or multiple, irregularly shaped nuclei with marginally condensed chromatin, and exhibited cell shrinkage, large clear vacuoles, and cytoplasmic blebbing, as well as intact cytoplasmic organelles, such as mitochondria and rough endoplasmic reticulum (Figures 5C to 5F).


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  		Figure 5.   Morphologic findings for anti-Fas antibody-induced apoptosis in bronchiolar epithelial cells. Control IgM (500 ng/ml)-treated cells (A) and CH-11 (500 ng/ml)- treated cells (B) were examined through phase contrast microscopy after 48 h incubation. Most of the cells were detached, and remaining attached cells became round in shape (B). Electron microscopic findings showed that bronchiolar epithelial cells treated with CH-11 (500 ng/ml) for 48 h reflected cell shrinkage (C; arrow), fragmented nuclei (D), marginal condensation of chromatin (E, F ), cytoplasmic blebbing (E; arrows), and large clear vacuoles (D, E; arrowheads). These findings are characteristic of apoptotic cells, which are distinct from relatively intact neighboring cells (C). (A, B; original magnification: × 200; bar in C, D, E, F : 1 µm).

IL-8 Secretion in Response to TNF-alpha and Fas Ligation

Figure 6a shows the relationship between IL-8 secretion and the concentration of CH-11 or TNF-alpha used to treat bronchiolar epithelial cells. This relationship was linear from a TNF-alpha concentration of 0.1 ng to 1 µg/ml and from a CH-11 concentration of 10 ng to 1 µg/ml, respectively. Although both TNF-alpha and Fas ligation led to IL-8 secretion, less IL-8 secretion was induced by Fas ligation than by TNF-alpha . Because the quantity of secreted IL-8 induced by 100 ng/ml CH-11 was equal to that induced by 4 ng/ml TNF-alpha , the experiments were done with these concentrations of CH-11 and TNF-alpha . The early kinetics of 100 ng/ml CH-11-induced IL-8 secretion were not significantly different from that of IL-8 secretion induced by 4 ng/ml TNF-alpha . Figure 6b shows the time-response relationship of both TNF-alpha and Fas ligation to IL-8 production. CH-11 and TNF-alpha induced IL-8 secretion with similar kinetics. This relationship was linear until 48 h.


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  		Figure 6.   IL-8 secretion in response to TNF-alpha and Fas ligation. (a) Concentration dependency. Bronchiolar epithelial cells were exposed to TNF-alpha or CH-11 (0.1-1,000 ng/ml). Supernatants were harvested after 24 h incubation for IL-8 measurement with an ELISA. (b) Time course. Cells were cultured with CH-11 (100 ng/ml) or TNF-alpha (4 ng/ ml) for indicated intervals. Supernatants were harvested for IL-8 measurement with ELISA. All experiments were repeated three times, and the figure shows the representative data from the three. Each point is the mean ± SE of three samples. Sensitivity of IL-8 ELISA was 20 pg/ml.

To determine the effect of IFN-gamma pretreatment on apoptosis and IL-8 secretion, the number of apoptotic cells and the quantity of IL-8 were measured after IFN-gamma pretreatment of cultures followed by treatment with TNF-alpha or CH-11. There was no significant difference in IL-8 secretion and apoptosis at 24 h with TNF-alpha or CH-11 alone as compared with IFN-gamma pretreatment followed by TNF-alpha or CH-11 (Figure 7). IFN-gamma alone did not induce apoptosis of bronchiolar epithelial cells (data not shown).


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  		Figure 7.   IL-8 secretion (left panel) and apoptosis (right panel) in response to TNF-alpha or Fas ligation with or without IFN-gamma pretreatment. Cells were exposed to TNF-alpha (4 ng/ml) or CH-11 (100 ng/ml) for 24 h with or without prior IFN-gamma (40 ng/ml) exposure for 6 h. Supernatants were harvested at 24 h for the measurement of IL-8 with an ELISA, and the quantification of apoptotic cells was done with flow cytometry. Results are shown as mean ± SE of three experiments.

To exclude cross-reactivity between TNF-alpha receptors and Fas, we used the monoclonal anti-Fas antibody ZB-4 in an attempt to block CH-11 ligation. ZB-4 was able to inhibit 73% of CH-11-mediated apoptosis (P < 0.01) and 87% of CH-11-mediated IL-8 secretion (P < 0.01) without a significant effect on TNF-alpha -mediated apoptosis or IL-8 secretion (Figure 8). ZB-4 alone did not induce apoptosis or IL-8 secretion (data not shown).


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  		Figure 8.   Effect of monoclonal anti-Fas antibody ZB-4 on CH-11- and TNF-alpha -mediated IL-8 secretion (left panel) or apoptosis (right panel). Bronchiolar epithelial cells were exposed to TNF-alpha (4 ng/ ml) or CH-11 (100 ng/ml) for 24 h in the presence or absence of ZB-4. ZB-4 was used at a concentration of 10 µg/ml and administered at 2 h before TNF-alpha or CH-11 administration. Supernatants were harvested at 24 h for measurement of IL-8 with an ELISA, and the measurement of the apoptosis rate was done simultaneously with flow cytometry. Results are shown as mean ± SE of three experiments. (* P < 0.01).

To study the requirement for new gene expression and protein synthesis in TNF-alpha - or Fas-mediated apoptosis and IL-8 secretion, we studied the effect of actinomycin D or cycloheximide. Figure 9 shows that actinomycin D or cycloheximide given concurrently with TNF-alpha or CH-11 neither enhanced nor diminished apoptosis. By contrast, secretion of IL-8 was inhibited when either actinomycin D or cycloheximide was given concurrently with TNF-alpha or CH-11 (P < 0.01). Thus, new gene expression and protein synthesis are required for TNF-alpha - or Fas-mediated IL-8 secretion, but are not necessary for TNF-alpha - or Fas-mediated apoptosis.


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  		Figure 9.   Effect of metabolic inhibitor on IL-8 secretion (left panel) or apoptosis (right panel). Bronchiolar epithelial cells were exposed to TNF-alpha (4 ng/ml) or CH-11 (100 ng/ml) for 24 h, either alone or in the presence of actinomycin D (1 µg/ml) or cycloheximide (10 µg/ml). Supernatants were harvested at 24 h for measurement of IL-8 with an ELISA, and the apoptosis rate was simultaneously measured with flow cytometry. Results are shown as mean ± SE of three experiments. (* P < 0.01.).

NF-kappa B Activation in Response to TNF-alpha or Fas Ligation

We focused our next set of experiments on the effects of TNF-alpha or Fas ligation on NF-kappa B activation. EMSA analysis revealed a rapid increase in NF-kappa B binding activity in the nuclear extracts from cells treated with each agonist (Figure 10). Nuclear extract harvested from untreated cells showed minor binding of a double-stranded oligonucleotide containing the consensus NF-kappa B recognition sequence. However, after treatment for 2 h with TNF-alpha (4 ng/ml) or CH-11 (100 ng/ml), the NF-kappa B-DNA binding activity increased. The specificity of the observed DNA- protein interaction was confirmed by the ability of excess unlabeled NF-kappa B oligonucleotide (a specific competitor) to inhibit binding. Addition of an antibody to the p65 component of NF-kappa B resulted in a retardation (supershift) of NF-kappa B-DNA binding activity. These data indicate that TNF-alpha or Fas ligation induces activation of NF-kappa B p65 hetero- or homodimers in bronchiolar epithelial cells.


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  		Figure 10.   Induction of transcription factor NF-kappa B by TNF-alpha or CH-11 as evaluated through EMSA of nuclear extracts. Cells were treated for 2 h with 500 ng/ml of control IgM (C), 4 ng/ml of TNF-alpha , or 100 ng/ml of CH-11; lanes 2 and 5: 125-fold unlabeled consensus NF-kappa B oligonucleotide added (specific competitor); lanes 3 and 6: preincubation of nuclear proteins with antibody directed against the p65 unit of NF-kappa B. The arrow indicates NF-kappa B-DNA binding activity, and the arrowhead indicates p65 component. Data are representative of three separate experiments.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We found that Fas ligation induced IL-8 secretion in addition to apoptosis in bronchiolar epithelial cells in vitro. Fas was expressed constitutively in bronchiolar epithelial cells, and was upregulated markedly by TNF-alpha with IFN-gamma pretreatment, but to only a small extent by TNF-alpha or IFN-gamma alone. Although TNF-alpha or Fas ligation induced apoptosis in a dose-dependent manner in bronchiolar epithelial cells, there was no significant increase in IL-8 secretion or apoptosis when IFN-gamma was administered before incubation with TNF-alpha or CH-11. Although another study suggested synergy between IFN-gamma and Fas ligation in the induction of apoptosis (32), IFN-gamma did not seem to act in synergy with the Fas-signaling pathway in inducing apoptosis or IL-8 secretion in bronchiolar epithelial cells.

To exclude cross-reactivity between TNF-alpha and Fas, we used a monoclonal anti-Fas antibody, ZB-4, to inhibit the binding of CH-11 to Fas. CH-11- but not TNF-alpha -mediated apoptosis and IL-8 secretion were significantly inhibited by ZB-4. Therefore, the respective binding of TNF-alpha to TNF-alpha receptor and of CH-11 to Fas was specific in our study.

Apoptosis in response to TNF-alpha or CH-11 did not require new gene expression or protein synthesis, suggesting the participation of an existing second messenger pathway in this process. However, IL-8 secretion induced by TNF-alpha or CH-11 did require new gene expression and protein synthesis. Recently, Abreu-Martin and colleagues showed that stimulation with CH-11 as well as TNF-alpha induced IL-8 secretion by a colon epithelial cell line (33). It has been reported that TNF-alpha induces IL-8 secretion by various cells (34), and that CD40-mediated stimulation also enhances IL-8 secretion by monocytes (37). TNF-alpha has been shown to induce IL-8 secretion by transactivation of the IL-8 gene via NF-kappa B activation. Several members of the TNF-alpha -/nerve growth factor-receptor family activate NF-kappa B through a common adapter protein, Traf2 (38, 39). Malinin and coworkers found a new protein with serine/ threonine kinase activity that binds to Traf2 and takes part in the activation of NF-kappa B by TNFRs and by Fas (23). In the present study, we found that Fas ligation activated NF-kappa B on bronchiolar epithelial cells. Therefore, Fas ligation may transactivate the IL-8 gene through NF-kappa B activation.

Despite similarities between cell-death signals transduced by CH-11 and TNF-alpha , the rapidity of apoptosis induced by Fas ligation distinguishes these two agonists from one another. Although both CH-11 and TNF-alpha killed approximately 23% of bronchiolar epithelial cells by 24 h, the number of apoptotic cells was significantly increased as early as 6 h after exposure to CH-11, but only at 24 h after exposure to TNF-alpha . These results suggest the existence of a signal-transduction pathway that is more efficient at causing apoptosis with Fas-binding than with TNF-alpha receptor binding. Other studies have also shown that Fas- derived signals lead to killing of most cells in a melanoma cell line within hours after stimulation, whereas TNFRp55- and TNFRp75-associated signals resulted in cell death after 2 to 3 d (40), and have shown the killing of colon epithelial cells (33) at 24 h after engagement of TNF-alpha .

We previously showed that the expression of Fas was upregulated in bronchiolar and alveolar epithelial cells, and that FasL was expressed in infiltrating lymphocytes in lung tissues from patients with IPF (7). The mouse model for IPF is bleomycin-induced pulmonary fibrosis, in which Fas and FasL were found to be upregulated in lung epithelial cells and infiltrating lymphocytes, respectively, and apoptosis of lung epithelial cells was detected (8). Therefore, the Fas/FasL pathway and excessive apoptosis of epithelial cells may be involved in the pathogenesis of pulmonary fibrosis. Furthermore, we demonstrated that inhalation of an agonistic anti-Fas antibody (Jo2) induced lung injury and fibrosis in mice (10). In this model, neutrophilic infiltration was seen in lung tissue after the inhalation of Jo2. Recently, Seino and associates showed that the introduction of complementary DNA (cDNA) for FasL into murine tumor cells did not affect the cells' growth in vitro, but caused their rejection in vivo. Neutrophils were primarily responsible for this rejection (11). Additionally, FasL expression on pancreatic islet cells results in massive neutrophilic infiltration and islet destruction (41, 42). These findings suggested a proinflammatory function of FasL. In the present study, Fas ligation induced apoptosis and IL-8 secretion in bronchiolar epithelial cells. IL-8 has been reported to induce changes in the shape of neutrophils, to activate the neutrophil respiratory burst, and to stimulate the release of oxygen free radicals and of elastase and other neutral proteases (43). As a potent chemoattractant for neutrophils, IL-8 may also contribute to the observed neutrophil influx into the pulmonary alveolus (44). We speculated that IL-8 secretion stimulated by the Fas ligation might be associated with neutrophilic infiltration and inflammation, and that it might participate in the pathophysiology of lung injury and pulmonary fibrosis as well as the excessive apoptosis induced by Fas ligation.

We found that in addition to inducing apoptosis in bronchiolar epithelial cells, Fas ligation induced IL-8 secretion by these cells. In addition, Fas ligation increased NF-kappa B-DNA binding activity. Because TNF-alpha activates the IL-8 promoter transcriptionally via NF-kappa B activation, IL-8 secretion induced by Fas ligation may be regulated by NF-kappa B activation. It has been shown that IL-8 levels are increased in BALF from patients with IPF (15, 16), and IL-8 appears to participate in the pathophysiology of IPF by sequestering and activating neutrophils. Therefore, induction of IL-8 in bronchiolar epithelial cells may lead to another pathogenic role of the Fas/FasL pathway in lung injury and pulmonary fibrosis.

    Footnotes

Abbreviations: enzyme-linked immunosorbent assay, ELISA; fluorescein isothiocyanate, FITC; interferon-gamma , IFN-gamma ; interleukin-8, IL-8; nuclear factor-kappa B, NF-kappa B; tumor necrosis factor, TNF; tumor necrosis factor receptor, TNFR.

(Received in original form April 20, 1998 and in revised form April 19, 1999).

Acknowledgments: This work was supported by a Grant-in-Aid for Scientific Research (09670620) from the Ministry of Education, Science, and Culture of Japan.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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K. Kuwano and N. Hara
Signal Transduction Pathways of Apoptosis and Inflammation Induced by the Tumor Necrosis Factor Receptor Family
Am. J. Respir. Cell Mol. Biol., February 1, 2000; 22(2): 147 - 149.
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S. Faouzi, B. E. Burckhardt, J. C. Hanson, C. B. Campe, L. W. Schrum, R. A. Rippe, and J. J. Maher
Anti-Fas Induces Hepatic Chemokines and Promotes Inflammation by an NF-kappa B-independent, Caspase-3-dependent Pathway
J. Biol. Chem., December 21, 2001; 276(52): 49077 - 49082.
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Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 1999 American Thoracic Society.