Induction of CD95 (Fas) and Apoptosis in Respiratory Epithelial Cell Cultures Following Respiratory Syncytial Virus Infection

Virology 257, 198–207 (1999) Article ID viro.1999.9650, available online at http://www.idealibrary.com on

Induction of CD95 (Fas) and Apoptosis in Respiratory Epithelial Cell Cultures Following Respiratory Syncytial Virus Infection D. R. O’Donnell, 1 L. Milligan, and J. M. Stark 2 Division of Pulmonary Medicine, Allergy and Clinical Immunology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039 Received October 23, 1998; returned to author for revision December 8, 1998; accepted February 10, 1999 Respiratory syncytial virus (RSV) infection is associated with epithelial cell death and vigorous inflammation. In mouse models, and in immunosuppressed patients, CD8 1 T cells are necessary for RSV clearance. In vitro, RSV has been shown to induce expression of several proteins on the respiratory epithelial cell, including RSV proteins, ICAM-1, and MHC class I, that can potentially interact with CD8 1 T cells in initiating apoptosis of the target cell. One mechanism of T-cell-directed cell death is the interaction of FasL on the CD8 1 T lymphocytes and Fas expressed on the target cell. In order to determine the ability of RSV to induce Fas on the respiratory epithelium, we studied the RSV infection of a human respiratory epithelial cell line (A549) in vitro. Fas mRNA and protein levels are increased two-to-fourfold following RSV infection, and transcriptional upregulation of Fas was demonstrated using promoter/reporter gene constructs. RSV infection directly resulted in cellular apoptosis, and the frequency of apoptotic cells was further increased by cross-linking with antibodies to Fas. These data demonstrate that RSV infection induces cellular apoptosis and suggest that interactions of surface Fas with T cells may further augment this process in vivo. © 1999 Academic Press Key Words: Fas; CD95; respiratory syncytial virus; apoptosis; NF-IL6.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory disease in infants. It is the primary virus isolated from infants with bronchiolitis, the most common reason for hospital admission under 1 year of age (Denny and Clyde, Jr., 1986; Anderson and Heilman, 1995). RSV infects respiratory epithelial cells and is associated with a vigorous inflammatory response resulting in epithelial cell death and viral clearance in vivo (Aherne et al., 1970; Taylor et al., 1984; Graham et al., 1988). Infection of cell lines and primary epithelial cell cultures with RSV lead to reproducible patterns of viral replication and histopathology ultimately leading to cell death (Collins, 1996; Stark et al., 1996; Noah and Becker, 1994). Recent studies in vitro using respiratory epithelial cells (primary cells or immortalized cell lines) have demonstrated that the respiratory epithelium is not just a passive barrier to viral invasion, but these cells actively release several cytokines and chemokines that can potentially modulate the nonspecific and immune responses to RSV infection, including interleukin-6 (IL-6),

IL-1, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), and RANTES (Noah and Becker, 1994; Fiedler et al., 1995; Patel et al., 1995; Becker et al., 1997; Saito et al., 1997). Moreover, RSV infection increases expression of cell receptors recognized by inflammatory cells, including ICAM-1 and MHC class I proteins (Stark et al., 1996). Therefore, the respiratory epithelium appears to play a pivotal role in the inflammatory responses to RSV and limiting its spread. Indeed, even in immunocompromised hosts RSV is rarely detected outside the lung, despite prolonged virus shedding (Cannon et al., 1988). Cell death can occur among the infected respiratory epithelial cells by two major mechanisms in eukaryotic cells: apoptosis and necrosis. Necrosis is a pathologic form of cell death that results from acute cellular injury and is characterized by rapid cell swelling and lysis (Thompson, 1995). Necrotic cell death could potentially result from cell membrane fusion caused by the viral F-protein or other direct viral toxicity. There is substantial evidence that apoptosis, or programmed cell death, results from a complex cascade of events possibly initiated by the activation of specific cellular kinases, leading to nuclear fragmentation (Vaux and Strasser, 1996; Anderson, 1997). Apoptosis could be triggered in RSV-infected cells directly by the virus or through “indirect” pathways activated by inflammatory cell attack of the virus-infected cells. In vivo, RSV infection is cleared, yet RSV disease is augmented by the cellular immune responses to the

1 Current address: Paediatrics, Imperial College of Science, Technology and Medicine, St. Mary’s Hospital, Norfolk Place, London W2 1PG UK. 2 To whom correspondence and reprint requests should be addressed at Division of Pulmonary Medicine, Allergy and Clinical Immunology, Children’s Hospital Medical Center, 3333 Burnet Avenue OSB5, Cincinnati, Ohio, USA 45229-3039. Fax: (513) 636 4615. E-mail: [email protected].

0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. RSV infection increases expression of Fas on respiratory epithelial cell cultures. A549 monolayers were exposed to media alone (a), UV-inactivated RSV (b), or active RSV (c) and Fas expression was measured 48 h following using FITC-conjugated murine anti-CD95 monoclonal antibody and flow cytometry. (A) is a flow cytometry tracing of a representative experiment showing uninfected cells, anti-Fas antibody (a, gray fill), treatment with UV-inactivated cells, anti-Fas antibody (b, dark fill), RSV-infected cells, anti-Fas antibody (c, solid line, no fill), and control cells, isotype-matched control antibody (d, no fill, dotted line). UV inactivation prevented Fas upregulation by the RSV preparations. (B) is a summary of the results of six separate experiments. Data are shown as median fluorescence for the experimental condition using anti-Fas antibody [individual experiments (closed circles) and their mean (open circles)] or isotype control antibodies [individual experiments (closed squares) and their mean (open squares)], n 5 6 per group. Error bars represent standard error of the mean; *P , 0.01 compared to uninfected and UV-inactivated controls.

virus (Cannon et al., 1988; Graham et al., 1991; Alwan et al., 1992, 1994; Tang and Graham, 1997). The inflammatory cells responding to infection could induce apoptosis of the RSV-infected epithelium through two separate apoptotic pathways—through degranulation and release of perforin and granzyme B and through direct cell–cell interactions between inflammatory cell Fas-L and Fas expressed on the target cell surface, resulting in activation of families of specific cellular proteases, including the caspases, that lead to cellular apoptosis (Vaux and Strasser, 1996; Anderson, 1997). Influenza and measles virus have been demonstrated to induce apoptotic cell death (Takizawa et al., 1993; Hinshaw et al., 1994; Esolen et al., 1995). Moreover, influenza virus was demonstrated to induce Fas gene expression in cultured Hela cells by activation of the nuclear transcription factor nuclear factor IL-6 (NF-IL6) (consensus sequences for this transcription factor have been demonstrated in the Fas promoter) (Wada et al., 1995). RSV also activates NF-IL6 following infection of respiratory epithelial cell monolayers, resulting in induction of IL-8 synthesis (Jamaluddin et al., 1996; Fiedler et al., 1996b). It was previously demonstrated that RSV infection induces expression of ICAM-1 and MHC class I molecules, making the RSV-infected respiratory epithelium a potential target for cytotoxic CD8 T cells and NK cells (Stark et al., 1996). In the present studies we report the effect of RSV infection on the expression of Fas by human respiratory epithelial cell (A549 cells) cultures. We demonstrate the upregulation of Fas protein and the induction Fas mRNA following RSV infection. Epithelial cell-expressed Fas is biologically functional: cross-linking of

Fas with monoclonal antibody results in apoptosis of RSV-infected but not control monolayers. Moreover, RSV infection alone induced significant levels of cellular apoptosis. These studies have importance in delineating the potential mechanisms that regulate inflammation, tissue injury, and, potentially, lung repair following RSV infection of the airway. RESULTS Induction of Fas expression on the cell surface after infection after RSV infection Uninfected A549 cells express low levels of Fas on the cell surface; however, following RSV infection there is significant increase in Fas levels (Fig. 1A). The upregulation of Fas requires exposure to live virus: A549 cells exposed to UV-inactivated virus express levels of Fas similar to that of mock-infected control cells (representative histograms, Fig. 1A). Figure 1B summarizes the results of six separate experiments, demonstrating the small but significant (P , 0.01) increase in Fas protein following RSV infection (consistently, approximately twofold higher in RSV-infected cells than in uninfected controls). Fas expression was increased by 6 h following RSV infection and increased only slightly thereafter (24–72 h, Fig. 2). Induction of Fas mRNA following infection with RSV Competitive PCR and Northern blot analyses were used to detect Fas mRNA in RSV-infected cells. Competitive PCR was performed using the MIMIC system

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FIG. 2. Time course of Fas expression following RSV infection of A549 cells. Fas expression on A549 cells was detected by flow cytometry using FITC-labeled specific murine monoclonal antibody to human Fas. The data show isotype controls (closed squares) and Fas (CD95) expression (closed circles). Fas expression increased significantly by 6 h and continued to increase through 72 h following RSV. The data are shown for no infection harvested at 72 h (C) and after infection at 6, 24, 48, and 72 h. The means and standard errors are shown at each time point; *P , 0.01 compared to uninfected controls.

(primers indicated under Materials and Methods). The major advantage of the MIMIC method is that the amplification irrelevant DNA is primed using the same primers (and therefore the same annealing conditions) as used for amplification of Fas. Using 30 cycles of PCR amplification, we consistently demonstrated a two- to fivefold increase in Fas following RSV infection compared to uninfected controls (data not shown). To confirm the PCR data, we performed Northern analysis for Fas mRNA, using purified, poly-adenylated mRNA. When 2–5 mg of poly(A) RNA was loaded onto the gels, both the 2.9- and 1.7-kDa Fas mRNA species were readily detected in both control and RSV-infected cells (using the 200-bp Fas probe amplified and 32 P-labeled using PCR, Materials and Methods) (Fig. 3). Fas mRNA was elevated in the RSV-infected cells compared to controls 24 h following RSV infection and remained elevated through 72 h (an approximately twofold increase by densitometry).

FIG. 3. Northern blot analysis of Fas mRNA from A549 cells following RSV infection. Poly-adenylated RNA was isolated from A549 cells (control or RSV infected) at intervals following RSV infection and was analyzed by Northern blot using a 32P-labeled probe to human Fas. Lane 1 represents an interferon-g-treated Fas-positive control. Lanes 2, 4, and 6 contain poly(A) RNA from control cells at 24, 48, and 72 h following mock infection. Lanes 3, 5, and 7 contain poly(A) RNA from RSV-infected cells at the same time points. Both 2.9- and 1.7-kDa Fas mRNA species are identified. Blots were probed for b-actin as a mRNA control for equal loading. Fas mRNA was present in greater amounts at all three time points compared to control (determined by densitometry).

lowing infection with RSV (Fig. 4). The analysis of the published sequence of Fas contains several potential NF-IL6 consensus binding sites. Previous reports that RSV infection activated NF-IL6 were confirmed under our experimental conditions using electrophoretic mobility shift assay (EMSA) and supershift assays (data not shown).

Transcriptional regulation of Fas following RSV exposure Because of the relatively small but consistent increases in the levels of both Fas protein and mRNA following RSV infection of A549 cells, analysis of Fas promoter activity was performed. For these studies, the Fas promoter was cloned into the vector, pGL2-basic, directing expression of the firefly luciferase reporter gene. The luciferase activity was modestly increased in A549 cells transfected with the Fas promoter construct. This activity was further increased (two- to fivefold) fol-

FIG. 4. Transcriptional upregulation of the Fas gene following RSV infection. The 1700 bp 59 proximal to the Fas gene (Fas promoter) was obtained by PCR amplification and cloned into pGL2Basic, directing expression of the firefly luciferase gene. The Fas–luciferase construct was cotransfected into A549 cells, along with a plasmid encoding b-galactosidase (to control for tranfection efficiency). Luciferase activity was determined 6 h following exposure to RSV and normalized to b-galactosidase. Unfilled bars indicate uninfected and hatched bars indicate RSV infected; *P , 0.01 compared to empty vector; †P , 0.01 compared to uninfected control.

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FIG. 5. RSV infection causes apoptosis in A549 cells, which can be further enhanced with antibody to Fas. Apoptotic cells were detected by Br-dUTP (BRDU) DNA strand break labeling, stained using FITC anti-BRDU antibodies, and analyzed by flow cytometry. (A–D) are representative flow cytometric plots; (A) is RSV infected 1 anti-Fas, (B) is RSV infected (no antibody), (C) is the uninfected control 1 anti-Fas, (D) is an uninfected control (no antibody). (E) is a summary of data from two separate experiments, n 5 4 per group: *P , 0.01 compared to control, †P , 0.01 compared to RSV infection in the absence of cross-linking antibody. Error bars represent standard error of the mean.

RSV induces apoptosis in A549 cells—role of Fas Because of the role of Fas in T-cell-induced apoptosis, we investigated the biologic activity of this protein using cross-linking monoclonal antibodies. Control cells exhib-

ited minimal levels of apoptosis with or without crosslinking of Fas (Figs. 5C and 5D, respectively). RSV infection alone increases apoptosis from less than 2% to greater than 10% of cells (Fig. 5B). Cross-linking Fas on

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RSV-infected cells with monoclonal antibody further enhanced apoptosis (Fig. 5A, P , 0.01). The results of four independent assays are summarized for clarity in Fig. 5E. DISCUSSION The experiments described here indicate that RSV infection increases the expression of CD95 (Fas) on respiratory epithelial cells. This upregulation was modest in quantity (about twofold) but is consistent in all measurements of Fas activation used (flow cytometry, Northern blot, competitive PCR, and promoter analysis) and consistent with levels of Fas activation following infection with influenza virus reported by Wada et al. (1995). One mechanism for the increased expression of Fas appears to be transcriptional upregulation mediated by RSV-induced activation of the transcription factor NFIL-6. RSV infection alone caused apoptosis in a human respiratory epithelial cell line (A549 cells) in culture. Moreover, Fas expressed on the infected cell surface is biologically functional—apoptosis could be further enhanced by cross-linking Fas on the infected cell surface, increasing levels of apoptosis in the RSV-infected cell monolayers. The Fas upregulation (both transcription and translation) parallels the increases in RSV mRNA, protein, and the production of infectious virus particles. The “kinetics” of RSV replication in A549 cells has been reported previously (Noah and Becker, 1994; Fiedler et al., 1995). Following exposure of A549 cells to RSV (m.o.i. 5 1), there is no mRNA for the RSV F protein (measured by Northern blot) detectable until 16–20 h postexposure. Infectious virus was present (using plaque assay) after 24 h and peaked by 72 h following infection (Fiedler et al., 1995). Virus replication is accompanied by increases in Fas (this report), IL-8 (Fiedler et al., 1995), ICAM-1 (Chini et al., 1998), and GM-CSF (Noah and Becker, 1994). Recently published studies demonstrate that type II alveolar cells display restricted expression of Fas (Fine et al., 1997). Fas mRNA was detected from isolates of primary rat type II and Fas expression on a subset of alveolar type II cells by in situ hybridization and immunohistochemistry in normal mouse lung (Fine et al.,1997). Our observations that the A549 cell line can express Fas is therefore consistent with in vivo observations. Viral replication is necessary for Fas production: UVinactivated RSV fails to upregulate Fas transcription or translation. Similarly, ribavirin treatment of A549 monolayers infected with RSV, or treatment of the cells with UV-inactivated virus, prevents IL-8 production (Fiedler et al., 1996a). How RSV upregulates expression of these proteins and the activation of a number of transcription factors is uncertain. For instance, RSV can increase ICAM-1 expression in respiratory epithelial monolayers through apocrine or paracrine effects of IL-1a induced in the virus-infected cells (Patel et al., 1995). In these stud-

ies, supernatants from virus-infected cells induced ICAM-1 expression, and this expression could be partially blocked by monoclonal antibodies to IL-1a. However, UV-inactivated virus was unable to induce Fas expression, implying that virus replication or transcription of viral proteins is necessary in the process. Clearly, much work remains to be done to define the mechanisms of viral-induced upregulation of cytokines (IL-8, GM-CSF) and cell surface proteins (Fas, ICAM-1, MHC class I), but these data will be key to our understanding of the regulation of host inflammatory responses to virus and viral clearance. A search of the Fas promoter revealed several possible factors involved in its upregulation by RSV. Several sites for NF-IL6 have been demonstrated in the 59 proximal DNA sequences of the Fas gene (Wada et al., 1995), and we demonstrate activation of NF-IL6 in RSV-infected A549. NF-IL6 induction is required for IL-8 upregulation following RSV infection (Fiedler et al., 1996b; Mastronarde et al., 1996; Jamaluddin et al., 1996) and was demonstrated by Wada et al. to contribute to Fas upregulation following influenza infection of Hela cells (Wada et al., 1995), where these investigators also found low-level induction of Fas following influenza infection. Interferon-g, a cytokine that induces Fas on many cell types, has not been found to be produced by A549 cells or respiratory epithelial cell cultures (unpublished data). The current studies demonstrate that the upregulation of NF-IL6 is temporally related to Fas upregulation, suggesting but not proving the role of NF-IL6 in RSV-induced Fas expression. In the present studies, we found that Fas was expressed at low levels by A549 cells and that RSV infection resulted in small but statistically significant increases in biologically active Fas. These small changes would be predictable because of the genetic organization of the Fas promoter (Wada et al., 1995). The promoter lacks TATA, GC, and CCAAT sequences, usually seen in promoters with higher transcription efficiencies. Moreover, there are no consensus sites for more “potent” nuclear transcription factors in the Fas promoter region, such as NF-kB and AP-1, transcription factors responsible for high-level induction of IL-8 and ICAM-1 following RSV infection (Wada et al., 1995; Fiedler et al., 1996b; Mastronarde et al., 1996; Chini et al., 1998). The Fas detected in the present studies was found to be biologically functional: cross-linking with monoclonal antibodies enhanced virus-induced apoptosis. Fine et al. were able to demonstrate Fas on freshly isolated alveolar type II cells and that cross-linking this protein resulted in apoptosis (Fine et al., 1997). Our present data are consistent with these published data on Fas upregulation and function in cells representative of the distal airway epithelium. Recently Takeuchi et al. demonstrated that RSV infection induced several caspases, including IL-1b converting enzyme (ICE) and its transcriptional activator interferon regulatory factor (IRF-1) (Takeuchi et

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al., 1998). ICE activation is proinflammatory in that it results in converting pro-IL-1b to active cytokine and indirectly contributes to an increase in IL-1a (Cohen, 1997). Therefore, the activation of ICE following RSV infection demonstrated by Takeuchi et al. (1998) is consistent with the findings by Patel et al., who demonstrated that RSV infection increased expression of IL-1a (Patel et al., 1995). Takeuchi also found that expression of CPP32 (caspase-3, one of the key executioner proteins of apoptosis (Cohen, 1997)) was enhanced following infection of A549 cells with RSV Long strain virus; however, they were not able to demonstrate either the apoptosis or the upregulation of cell surface Fas expression that we demonstrate in the studies presented here. The difference in ability to demonstrate apoptosis in the present study and that of Takeuchi may reflect differences in virus virulence, as found with Sendai virus infection. Itoh et al. used studied Sendai virus strains with different virulence (Itoh et al., 1998): a strain of low virulence greatly enhanced apoptosis and was associated with less lung pathology in mice compared to more virulent strains. Differences in virus virulence, local differences in the A549 lines, or differences in methodology used to determine apoptosis may account for the difference in virus-induced apoptosis reported by Takeuchi and the present report. It is possible that RSV induces cell death by apoptosis or other pathways under slightly different experimental conditions. However, the data reported here are consistent with reports of virus-induced apoptosis by Sendai virus (Itoh et al., 1998), measles (Esolen et al., 1995), and influenza viruses (Wada et al., 1995) and demonstate that under the experimental conditions used, RSV induces Fas expression and causes cellular apoptosis. The relative importance of each pathway (direct cytopathology and necrosis vs apoptosis) in RSV infection is unclear. Apoptosis of the infected cell (through direct effects of virus or through activation of Fas by lymphocytes) would potentially provide a host animal with a means to limit virus replication while limiting the “collateral damage” in the airway (Collins, 1995). Apoptosis is a physiologic mechanism of cell death. Recent studies demonstrate that virus infection and viral gene products can specifically activate or inhibit apoptosis from occurring (Collins, 1995). Apoptosis of infected cells could help to limit the replication of virus and would therefore be a disadvantage to the infecting virus. Indeed, several viruses produce products that specifically prevent the apoptotic process, allowing greater viral replication or viral survival (Collins, 1995). How RSV induced apoptosis in our studies is unknown, but likely involved initiation of the apoptotic cascade through mechanisms other than direct activation of Fas, since apoptosis was further enhanced by cross-linking Fas using monoclonal antibodies. Apoptotic cell death induced through CD95 ligation is believed to be mediated by a mechanism consisting of a

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series of cysteine proteases (caspases) that are activated as a hierarchical cascade, resulting in irreversible regulated termination of all vital cell processes (Vaux and Strasser, 1996; Barinaga, 1998; Anderson, 1997). Although a great deal of information is known about CD95mediated lymphocyte apoptosis, relatively little is known about the role of CD95 in epithelium and the role of epithelial cells in the induction of specific inflammatory and immune responses. Recent studies provide evidence that the respiratory epithelium has the ability to initiate or perpetuate lung inflammatory responses to RSV infection. In vitro and in vivo studies demonstrate that the RSV-infected respiratory epithelial cells secrete a number of potent proinflammatory molecules, including cytokines (IL-8, RANTES, GM-CSF), that can contribute to inflammatory cell recruitment and activation (Noah and Becker, 1994; Fiedler et al., 1995; Becker et al., 1997). In addition, the RSV-infected respiratory epithelial cells express increased levels of receptor proteins potentially recognized by the immune system, including ICAM-1, MHC class I, and Fas, recognized by migrating inflammatory cells, potentially directing and retaining them in sites of injury. RSV-induced apoptosis alone cannot limit RSV infection in the lung. Irradiated mice can be persistently infected with RSV: this infection can be cleared by passive transfer of CTL (Cannon et al., 1987). Likewise, RSV infection (pneumonia) is associated with significant mortality rates in immunocompromised human patients (Hall et al., 1986; Englund et al., 1988; Harrington et al., 1992). Many laboratories have studied the role of T lymphocytes in this process and have demonstrated the central role of cytotoxic T cells in clearing virus infection and in virus-induced lung inflammation (Openshaw, 1995; Graham, 1995). CTL-induced killing of virus-infected respiratory epithelial cells is mediated by granular proteins (perforin and granzyme B) and through FasL interactions with Fas on the target cell. Our data demonstrate the presence of all the key components for CD8 T cell–infected epithelial cell interactions. We have previously demonstrated that RSV increases expression of the RSV glycoproteins ICAM-1 and MHC class I antigen on the surface of RSV- infected respiratory epithelial cells (Stark et al., 1996), which would allow interactions with CD8 T lymphocyte through the TCR and b2 integrins (Springer, 1995). FasL–Fas interactions could then mediate signaling for apoptosis of the infected epithelial target. The relative importance of these mechanisms in RSV infection remains to be determined, but clearly, the ability to regulate the extent of these inflammatory responses is key to understanding and treating the immunopathology of RSV lower respiratory disease. In summary, we show for the first time that RSV induces apoptosis and Fas expression in the A549 pneumocyte cell line. Fas expression is enhanced and allows activation of the apoptotic cascade by cross-linking antiFas antibody. These effects are temporally associated

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with activation of the transcription factor NF-IL6. Apoptosis may play a role in modulating airway inflammation caused by RSV and eliminating virus from the lung. MATERIALS AND METHODS Reagents Tissue culture media and reagents, including incomplete Hanks’ balanced salt solution (iHBSS), Eagle’s minimal essential medium (MEM), trypsin (0.5%), and fetal calf serum (FCS) were obtained from GIBCO BRL (Grand Island, NY). Plasticware (pipettes, flasks, tissue culture dishes, Falcon) were obtained from Fisher Scientific. Human respiratory epithelial cell (REC) culture Due to its low baseline expression of ICAM-1, A549, an immortalized human alveolar type 2 epithelial cell line (ATCC, Rockville, MD), was selected as a source of REC for these studies (Lieber et al., 1976). A549 cells were maintained in MEM supplemented with 8% FCS in ventilated 150-cm 2 sterile tissue culture flasks (Corning, Corning, NY). When confluent, the cells were treated with trypsin–EDTA and passed into sterile 24-well tissue culture plates (Falcon) at a density of 50,000 cells/cm 2. The A549 cultures used for these studies were limited to passage numbers 82–95. Cell lines and virus were tested for mycoplasma prior to the studies described. Propagation of virus, UV inactivation, and infection of A549 cultures Respiratory syncytial virus (RSV A2) (ATCC) was propagated in near confluent monolayers of Vero cells (African green monkey kidney, ATCC) grown in sterile tissue culture plates (20 3 100 mm) as described previously (Fiedler et al., 1996b). An aliquot of the virus preparation was titrated by plaque assay on CV1 (African green monkey kidney, ATCC) cells grown in 24-well tissue plates. UV inactivation of an aliquot of RSV stock virus was performed by transillumination of the RSV preparation in a sterile cell culture dish for 20 min, as previously described (Fiedler et al., 1995). Infecting susceptible CV1 cells with a volume of UV-irradiated virus preparation that should have contained 10 7 PFU of RSV resulted in no detectable infectious virus, confirming that the virus had been rendered nonreplicative. “Infections” with the UVtreated RSV were done in parallel with a nonirradiated aliquot of virus, and the volume of UV-inactivated virus used for “infection” was based on the volume of nontreated preparation needed to give the desired multiplicity of infection. Flow cytometric analysis Flow cytometry was used to identify Fas (CD95) on control and RSV-infected A549 cells. Near confluent

monolayers were exposed to RSV at an estimated m.o.i. 5 1, and the infected cells (and uninfected controls) were incubated at 37°C until harvested for study. Cells were removed from the culture dishes using 10 mM EDTA as described previously (Stark et al., 1996), fixed using 2% formaldehyde in hypertonic PBS for 20 min, and resuspended in PBS containing 1% BSA and 0.1% sodium azide (PBS/BSA/azide). The A549 cells (5 3 10 5) were stained for 20 min with either FITC-conjugated anti-human CD95 (mouse IgG1 isotype, Pharmingen, San Diego, CA) or isotype-matched FITC-conjugated control at 12.5 mg/ml. Flow cytometric data were acquired using a FACScan Flow Cytometer (Becton–Dickinson, San Jose, CA) and analyzed using CELLQuest software (Becton–Dickinson). RNA isolation and Northern blot analysis Fas mRNA production following RSV infection was analyzed by Northern blot using standard procedures, modified as described previously (Chomoczynski and Sacchi, 1987; Sambrook et al., 1989; Fiedler et al., 1996b; Chini et al., 1998) using the Qiagen RNeasy kit following the manufacturer’s protocols (Qiagen, Chatsworth, CA). It was necessary to prepare poly(A) RNA using the PolyATtract mRNA Isolation System, following the manufacturer’s directions (Promega, Madison, WI). For Northern blot analysis of these samples, 2 mg of poly(A) RNA was separated by electrophoresis in glyoxal–agarose gels and transferred to a nylon membrane (Hybond N, Amersham Life Science, Arlington Heights, IL). The blots were probed with labeled cDNAs corresponding to Fas (200-bp PCR product, below) or b-actin labeled with 32P, using a random primer labeling kit (Gibco/BRL, Gaithersburg, MD). Blots were stripped between hybridizations with each probe in 0.013 SSPE/0.1% sodium dodecyl sulfate (SDS) at 95°C for 30 min (Sambrook et al., 1989). Cloning of the Fas gene The Fas gene was cloned by PCR using oligonucleotides determined by the published sequence. (Fas 59 primer: 59-ATG CTG GGC ATC TGG ACC CTC-39; Fas 39 primer: 59-CTA GAC CAA GGT TTG GAT TTC-39). The PCR product was purified from low melting point agarose (Gibco/BRL) using the Elutip method (Schleicher & Schuell Inc., Keene, NH). This PCR fragment (1 kb) was then cloned into the pGEM-T vector using the manufacturer’s directions (Promega, Madison, WI), and appropriate constructs were identified by restriction endonuclease analysis and by PCR using nested internal primers. The Fas gene products were subcloned into the BlueScript(KS) (Strategene, La Jolla, CA) vector using the PstI and ApaI sites. The identity of the cloned PCR product was confirmed by two methods: PCR analysis using a nested set of PCR primers (59 internal primer: ATG CTG GGC ATC TGG ACC CTC-39; 39 internal primer: AGC TTT CCT TTC ACC TGG AGG-39) and DNA sequence analysis

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of 100 bases at each end of the putative gene product (confirming sequence and orientation, comparing the product to published Fas sequence (Itoh et al., 1991; Wada et al., 1995)). Cloning of the 59-flanking region of the Fas gene (the Fas promoter) The Fas promoter was cloned by PCR amplification using human genomic DNA as the template. Primers were synthesized using published sequence by the University of Cincinnati DNA Core Facility (59 primer containing a BglII site: 59-GTG GAA GAT CTG GTT GTT GAG CAA TCC TCC GAA GTG-39; 39 primer containing a BglII site: 59-CCC GGG GTA CCA AGC TTT TTT GGC TAC ATT TTT TTA-39). PCR was performed at a 60° annealing temperature, and the reaction optimized by supplementing with 5% DMSO and 10 mM MgCl 2. The expected PCR product (approximately 1.7 kb) was purified using electrophoresis through low melting point agarose and elution as described above and cloned into pBlueScript(KS) at the EcoRV site after adding unpaired T’s. For the expression experiments, this promoter construct was subcloned into pGL2-basic (Promega) using the BglII and KpnI restriction sites added by PCR. The resulting product and its orientation were confirmed by limited DNA sequence analysis (200 bp from each end). Competitive PCR studies Because of the low abundance of Fas mRNA in the A549 cultures, competitive PCR studies were performed to quantify Fas message following infection with RSV. The competitive PCR experiments were performed using the Clontech MIMIC Construction Kit according to the manufacturer’s instructions (Clontech Laboratories Inc., Palo Alto CA). The PCR primers for the neutral MIMIC cDNA were constructed to contain the Fas PCR primers at its ends, yielding a fragment of approximately 400 bp (59 primer: 59-ATG CTG GGC ATC TCC ACC CTC CGC AAG TGA AAT CTC CTC CG-39; 39 primer: 59-AGC TTT CCT TTC ACC TGG AGG TCT GTC AAT GCA GTT TGT AG-39). PCR products were separated using electrophoresis in 1–2% agarose and identified by staining with ethidium bromide. The resulting DNA bands were quantified using a phosphorimager, comparing intensities with the MIMIC competitive products, as described by the manufacturer. Fas promoter activation/transfection experiments In order to test the hypothesis that RSV upregulation of Fas gene expression occurs at the transcriptional level, we utilized the reporter construct described above. A549 cells were transfected with the reporter construct and pCMV/pCMV/b-galactosidase as described previously (Chini et al., 1998; Fiedler et al., 1996b). Following transfection, A549 cells were incubated overnight prior to

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infection with RSV. Infections were performed as described above, and samples were obtained at intervals for measurement of luciferase and b-galactosidase activity as described previously (Chini et al., 1998; Fiedler et al., 1996b). Preparation of nuclear extracts and EMSA for NF-IL6 Nuclear extracts were prepared as described previously (Fiedler et al., 1996b). The nuclear extracts were aliquotted and stored at 270°C until assay. EMSA were performed using the nuclear extracts, and 32P-labeled double-stranded oligonucleotides containing the NF-IL6 consensus sequence (CAG TTG GCA AAT CGT, prepared by the Oligonucleotide Core Facility at the University of Cincinnati) (Akira et al., 1990). Equivalent amounts of protein (milligram basis) were incubated with the labeled probe (100,000 dpm) for EMSA studies. For antibody supershift assays, 1 mg of rabbit polyclonal antibody to NF-IL6 (Santa Cruz Biotechnology, Santa Cruz, CA) was incubated on ice for an additional 60 min. Bound and free probes were resolved using nondenaturing polyacrylamide gel electrophoresis (Fiedler et al., 1996b). Cross-linking Fas with anti-CD95 monoclonal antibody In order to assess the biologic function of the Fas detected on the A549 cell surface, cross-linking studies were performed using antibody to CD95. Anti-CD95 (azide free, clone DX2 mouse isotype IgG1 (Pharmingen) and Protein G (recombinant from Streptococcus sp., Sigma Chemical Co. St. Louis, MO) were present in the A549 cultures from the time of RSV infection until harvesting (72 h), at concentrations of 5 and 2 mg/ml, respectively. Apoptosis studies A549 cells were analyzed for apoptosis using the ApoBRDU kit (Pharmingen) and flow cytometry. Cells were harvested for apoptosis studies by gentle treatment with trypsin. Supernatants from each well were saved so that detached cells would be included in the analysis. Acridine orange staining was performed at 5 mg/ml for 15 min and cells were visualized by ultraviolet microscopy. Further cell aliquots were fixed with 1% paraformaldhyde for 15 min and then 70% ethanol at 220°C overnight. Cells were then washed and labeled with BRDU before staining with FITC-conjugated antiBRDU and treatment with propidium iodide and RNase (ApoBRDU kit, Pharmingen). Cells were then analyzed by flow cytometry. Data were collected by gating on propidium iodide and expressed as percentage FITC positive by comparison with known controls. Statistical analysis Data are expressed 6 standard deviation. Statistical analysis of the data included an overall ANOVA followed

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by paired Student’s t tests, using the statistical functions in the Quattro Pro spreadsheet program (Corel Corp., Farmingdale, NY). A probability of P , 0.05 was considered significant. Bonferroni adjustments were used to correct for multiple comparisons. ACKNOWLEDGMENTS The authors are grateful to Kara Dolries and Dr. Michael Fiedler for the kind gift of some of the RSV stocks used in these studies, to Susan McDowell for help in preparation of the manuscript, and to Dr. R. Hirsch for generous use of a FACScan flow cytometer. This research was supported in part by the American Lung Association and the Cystic Fibrosis Foundation.

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