Bafilomycin A1 inhibits rhinovirus infection in human airway epithelium: effects on endosome and ICAM-1

Tomoko Suzuki1, Mutsuo Yamaya1, Kiyohisa Sekizawa2, Masayoshi Hosoda1, Norihiro Yamada1, Satoshi Ishizuka1, Katsutoshi Nakayama1, Masaru Yanai1, Yoshio Numazaki3, and Hidetada Sasaki1

1 Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai 980-8574; 3 Virus Center, Clinical Research Division, Sendai National Hospital, Sendai 983-0045; and 2 Department of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba 305-8575, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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To examine the effects of bafilomycin A1, a blocker of vacuolar H+-ATPase, on rhinovirus (RV) infection in the airway epithelium, primary cultures of human tracheal epithelial cells were infected with RV14. Viral infection was confirmed by showing that viral RNA in the infected cells and the viral titers in the supernatants of infected cells increased with time. RV14 infection upregulated the production of cytokines and mRNA of intercellular adhesion molecule (ICAM)-1 in epithelial cells. Bafilomycin A1 reduced the viral titers of RV14 and inhibited the production of cytokines and ICAM-1 before and after RV14 infection. Bafilomycin A1 reduced susceptibility of epithelial cells to RV14 infection. RV14 increased activated nuclear factor-kappa B in the cells, and bafilomycin A1 reduced the activated nuclear factor-kappa B. Bafilomycin A1 decreased the number of acidic endosomes in the epithelial cells. These results suggest that bafilomycin A1 may inhibit infection by RV14 by not only blocking RV RNA entry into the endosomes but also reducing ICAM-1 expression in the epithelial cells. Bafilomycin A1 may therefore modulate airway inflammation after RV infection.

intercellular adhesion molecule-1; asthma; common cold; airway inflammation; vacuolar adenosine 5'-triphosphatase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PERSPECTIVE STUDIES HAVE INDICATED that asthma attacks are associated with infections of various viruses including rhinoviruses (RVs) and respiratory syncytial viruses (18, 24). Studies using PCR-based diagnostics have emphasized the importance of RVs by demonstrating that RVs are responsible for 80-85 and 45% of the asthma flairs in 9- to 11-yr-old children and adults, respectively, with RV being the most commonly implicated pathogen (18, 24). In contrast to a variety of other respiratory pathogens (e.g., influenza virus and adenovirus), cell cytotoxicity does not appear to play a major role in the pathogenesis of RV infection (32), but the clinical and pathological features of RV infection are, to a great extent, due to the elaboration of a variety of inflammatory mediators by the host (47).

The vacuolar H+-ATPases (V-ATPases) are a family of ATP-driven proton pumps responsible for the acidification of a variety of intracellular compartments in eukaryotic cells (10). V-ATPases provide the acidic environment required for dissociation of internalized ligand-receptor complexes within endosomes, for activating the secreted lysosomal enzymes, for facilitating protein processing and degradation by acid-dependent proteases, and for assisting the degradation of the bone matrix (10). Furthermore, exposure of influenza virus to a low pH within the endosomes by V-ATPases induces the formation of a fusion pore between the viral and endosomal membranes that permits entry of the viral RNA (40). The specific V-ATPase inhibitor bafilomycin A1 (5) blocks the infection of influenza virus and RV in HeLa cells and Madin-Darby canine kidney (MDCK) cells (3, 26, 28) and inhibits the uncoating of RV type 2 and type 14 (RV14) from late endosomes (2, 29). However, the role of V-ATPases in RV infection in human airway epithelial cells, the primary target for respiratory viruses, has not been elucidated.

Infection of epithelial cells with RV induces production of several cytokines such as interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-alpha , and granulocyte-macrophage colony-stimulating factor (34, 36). These cytokines are known to mediate a wide variety of proinflammatory and immunoregulatory effects (1) and may play an important role in the pathogenesis of RV infections. Acidic pH with V-ATPases appears to play a role in the production of proteins in the cells (42). However, the effects of V-ATPase inhibitors on the production of inflammatory mediators and the major RV receptor, intercellular adhesion molecule-1 (ICAM-1), in RV infection have not been investigated (27, 36, 47).

We therefore investigated whether inhibitors of V-ATPases have inhibitory effects on RV infection in cultured human tracheal epithelial cells and determined the mechanisms responsible for the inhibition of RV infection. We also studied the effects of V-ATPase inhibitors on the production of cytokines and ICAM-1 by RV infection.


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Medium components. Reagents for the cell culture medium were Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, fetal calf serum (FCS), gamma -globulin-free fetal calf serum (GGFCS; all from GIBCO BRL, Life Technologies, Palo Alto, CA), trypsin, EDTA, dithiothreitol, Sigma type XIV protease, human placental collagen, penicillin, streptomycin, gentamicin, amphotericin B (all from Sigma, St. Louis, MO), Ultroser G serum substitute (USG; BioSepra, Marlborough, MA), 5-(N-ethyl-N-isopropyl)amiloride (EIPA; Molecular Probes, Eugene, OR), and bafilomycin A1 (Wako Pure Chemical Industries, Osaka, Japan).

Human embryonic fibroblast cell culture. Human embryonic fibroblast cells were cultured in MEM containing 10% FCS supplemented with 5 × 104 U/l of penicillin and 50 mg/l of streptomycin in a Roux-type bottle (Iwaki Glass, Chiba, Japan) sealed with a rubber plug (25). Confluence was achieved at 7 days, at which time the cells were collected by trypsinization (0.05% trypsin and 0.02% EDTA). The cells (1.5 × 105 cells/ml) suspended in MEM containing 10% FCS were then plated in glass tubes (15 × 105 mm; Iwaki Glass) that were sealed with rubber plugs and cultured at 37°C.

Human tracheal epithelial cell culture. Tracheae for cell culture were obtained 3-6 h after death from 80 patients (age 63 ± 4 yr; 39 women and 41 men) under a protocol passed by the Tohoku University (Sendai, Japan) Ethics Committee. Forty of the patients were smokers. None had a respiratory illness including bronchial asthma, and they died of acute myocardial infarction (n = 22), congestive heart failure (n = 6), malignant tumor other than lung cancer (n = 23), rupture of aortic aneurysm (n = 5), liver cirrhosis (n = 3), renal failure (n = 4), leukemia (n = 5), malignant lymphoma (n = 3), cerebral bleeding (n = 6), and cerebral infarction (n = 3).

Isolation and culture of the human tracheal surface epithelial cells were performed as previously described (44). In brief, the surface epithelium was scored into longitudinal strips and pulled off the submucosa. The tracheal surface epithelial cells were isolated by digestion with protease (0.4 mg/ml; Sigma type XIV) dissolved in phosphate-buffered saline (PBS) at 4°C overnight. The cells were pelleted (200 g for 10 min) and suspended in DMEM-Ham's F-12 medium (50:50 vol/vol) containing 5% FCS. Cell counts were made with a hemocytometer, and estimates of viability were done with trypan blue and by measuring the amount of lactate dehydrogenase in the medium as previously reported (36). Protein concentrations were measured with the method previously reported (44). The cells were then plated at 5 × 105 viable cells/ml (2 × 105 cells/cm2) in round-bottom glass tubes (15-mm diameter and 105-mm length; Iwaki Glass) coated with human placental collagen (44). The glass tubes were sealed with rubber plugs, kept stationary at a slant of ~5°, and cultured at 37°C. This medium was replaced by DMEM-Ham's F-12 medium containing 2% USG on the first day after the cells were plated. The cell culture medium was supplemented with 105 U/l of penicillin, 100 mg/l of streptomycin, 50 mg/l of gentamicin, and 2.5 mg/l of amphotericin B. We used primary cultures of human tracheal epithelial cells on days 6-14 of culture for the experiments.

We confirmed cilia beating on the epithelial cells and the absence of fibroblasts in glass tubes using an inverted microscope (MIT-2, Olympus, Tokyo, Japan) from the beginning of the cell culture to the end of the experiments. Furthermore, to determine whether cultured cells can form tight junctions, we performed parallel cultures of human tracheal epithelial cells on Millicell CM inserts (0.45-µm pore size and 0.6-cm2 area; Millipore Products Division, Bedford, MA) to measure electrical resistance and short-circuit current with Ussing chamber methods (44). When the cells cultured under these conditions become differentiated and form tight junctions without contamination by fibroblasts, they have values of >40 Omega  · cm2 for resistance and >10 µA/cm2 for short-circuit current (44). Therefore, cultured human tracheal epithelial cells were judged as cells able to form tight junctions and were used for the following experiments when cells on the Millicell CM inserts had a high resistance (>40 Omega  · cm2) and a high short-circuit current (>10 µA/cm2). We observed whether the human tracheal epithelial cells made a dome formation to confirm that cells on solid glass supports form tight junctions, and we found that the human tracheal epithelial cells make a dome formation when the cells made confluent cell sheets on days 5-7 of culture as described by Widdicombe et al. (41).

To further exclude fibroblast contamination, the cells were examined with cytokeratin staining. The cells cultured in glass tubes were collected by trypsinization and cultured on glass slides overnight at 37°C in a CO2 incubator. The cells were then fixed with 4% paraformaldehyde and 0.05% Triton X-100 in 0.1 M phosphate buffer for 15 min at room temperature. After being rinsed with PBS, the cells were incubated with 10% skim milk to block nonspecific binding of the first and second antibodies. The sections were overlaid with either LeukoStat anti-keratin antibody (MAK-5, Triton, Alameda, CA), anti-vimentin antibody (DAKO-VIMENTIN, DAKO, Santa Barbara, CA), or an isotype-matched mouse monoclonal antibody with an irrelevant specificity (Chemicon International) in PBS containing 1% bovine serum albumin. Anti-keratin and anti-vimentin antibodies label epithelial cells and fibroblast cells, respectively (11, 33). After an overnight incubation at 4°C and a wash with PBS, the sections were incubated with biotinylated horse anti-mouse immunoglobulins (Vector Laboratories) diluted with PBS for 1 h at room temperature followed by incubation with the avidin-biotin-peroxidase complexes (Vectastain ABC kit, Vector Laboratories) for 1 h at room temperature. The sections were developed by exposure of the substrate to 3,3'-diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan) and were counterstained with methyl green.

We found from these experiments that nearly all the cells (>95%) from the monolayers of human tracheal epithelial cells in the tubes reacted with the anti-keratin antibody, but they did not react with the anti-vimentin antibody (data not shown). As a positive control, we stained the human embryonic fibroblast cells with anti-vimentin antibody and obtained positive signals (data not shown).

Viral stocks. RV14 was prepared in our laboratory from patients with common colds (25). RV can be distinguished from enterovirus, which also produces "entero-like" cytopathic effects on human embryonic fibroblasts, by inactivation of RV after treatment with acid (25). Furthermore, RV14 was identified with a microneutralization test with an antibody for RV14 as previously described (14). Stocks of RV14 were generated by infecting human embryonic fibroblast cells cultured in glass tubes in 1 ml of MEM supplemented with 2% GGFCS, 5 × 104 U/l of penicillin, and 50 mg/l of streptomycin at 33°C. The cells were incubated for several days in glass tubes in 1 ml of MEM supplemented with 2% GGFCS until the cytopathic effects were obvious, after which the cultures were frozen at -80°C, thawed, and sonicated. The virus-containing fluid so obtained was frozen in aliquots at -80°C. The content of the viral stock solutions was determined with the human embryonic fibroblast cell assay described in Detection and titration of viruses.

Detection and titration of viruses. RVs were detected by exposing confluent human embryonic fibroblast cells in glass tubes to serial 10-fold dilutions of virus-containing medium in MEM supplemented with 2% GGFCS. The glass tubes were then incubated at 33°C for 7 days, and the cytopathic effects of the viruses on the human embryonic fibroblast cells were observed with an inverted microscope (MIT, Olympus) as previously reported (25). The amount of specimen required to infect 50% of the human embryonic fibroblast cells [50% tissue culture infectious dose (TCID50)] was determined.

We examined in the preliminary experiments whether either a V-ATPase inhibitor, bafilomycin A1, or blockers of Na+/H+ exchangers (NHEs), EIPA and FR-168888, affect RV14 titration in human embryonic fibroblasts. We performed titration of RV14 using DMEM-Ham's F-12 medium containing 2% USG and RV14 at concentrations ranging from 101 to 105 TCID50 units/ml supplemented with either bafilomycin A1 (0.1 µM) (22), EIPA (10 µM), or FR-168888 (10 µM; a gift from Fujisawa Pharmaceutical, Tokyo, Japan) (6, 43). The medium containing RV14 supplemented with either bafilomycin A1 (0.1 µM), EIPA (10 µM), or FR-168888 (10 µM) was diluted 10-fold with MEM supplemented with 2% GGFCS and added to the human embryonic fibroblasts in glass tubes to observe the cytopathic effects. We found that neither bafilomycin A1 (0.1 µM), EIPA (10 µM), nor FR-168888 (10 µM) influenced the cytopathic effects on human embryonic fibroblasts at any content of RV14.

Viral infection of human tracheal epithelial cells. The medium was removed from the confluent monolayers of human tracheal epithelial cells and replaced with 1 ml of DMEM-Ham's F-12 medium containing 2% USG. The human tracheal epithelial cells were infected with RV14 by exposure to culture supernatants of human embryonic fibroblasts infected with RV14. As a mock infection, epithelial cells were exposed to the supernatants from fibroblasts cultured in medium alone. In the preliminary experiments, we found that the culture supernatants of human embryonic fibroblasts without RV14 infection did not contain significant amounts of IL-1beta and TNF-alpha and that they did not alter the levels of IL-1beta and TNF-alpha in the supernatants of cultured human tracheal epithelial cells from baseline. RV14 was added at a concentration of 105 TCID50 units/ml. After a 1-h incubation at 33°C, the viral solution was removed, and the cells were rinsed one time with 1 ml of PBS. The cells were then placed in DMEM-Ham's F-12 medium containing 2% USG supplemented with 105 U/l of penicillin, 100 mg/l of streptomycin, 50 mg/l of gentamicin, and 2.5 mg/l of amphotericin B and cultured at 33°C with rolling in an incubator (HDR-6-T, Hirasawa, Tokyo, Japan) (36). To measure the time course of viral release for the first 24 h, we used four separate cultures from each trachea, and the culture supernatants were collected 1, 6, 12, or 24 h after RV14 infection. Furthermore, to measure the viral release for 1-3, 3-5, or 5-7 days, the human tracheal epithelial cells cultured in the tubes were rinsed with PBS, and fresh DMEM-Ham's F-12 medium containing 2% USG was added at 24 h after RV14 infection. The whole volume of the medium was then taken for the measurement of viral content, and the same volume of fresh medium was replaced on days 3 and 5, and the whole volume of the medium was taken on day 7. The supernatants were stored at -80°C for the determination of viral content. Furthermore, cell-associated viral content was analyzed with sonicated human tracheal epithelial cells in MEM. The viral content in the supernatant and the cell-associated viral content are expressed as TCID50 units per milliliter and TCID50 units per 106 cells, respectively (36).

Cytokine assays. Because RV14 infection increased the production of various cytokines from primary cultures of human tracheal epithelial cells (36), we measured IL-1alpha , IL-1beta , IL-6, IL-8, TNF-alpha , interferon (IFN)-alpha , IFN-beta , and IFN-gamma by specific enzyme-linked immunosorbent assays (ELISAs). Sensitivities of the assays were 25 pg/ml for the IFN-alpha ELISA kit (COSMO BIO, Tokyo, Japan); 10 pg/ml for the IL-1alpha , IL-1beta (both from Ohtsuka, Tokushima, Japan), IL-6, and IL-8 (both from Toray, Tokyo, Japan) ELISA kits; 4 pg/ml for the TNF-alpha ELISA kit (Ohtsuka); 3 pg/ml for the IFN-gamma ELISA kit (Genzyme); and 1 U/ml for the IFN-beta ELISA kit (BioSource International, Camarillo, CA). In preliminary experiments, we found that the concentration of TNF-alpha in the culture medium was low (0-10 pg/ml). Therefore, we concentrated the culture medium by freeze-dry methods with a centrifugal vaporizer (Tokyo Rikakikai, Tokyo, Japan) before measuring the concentration of TNF-alpha . After the culture medium was freeze-dried, the pellet was dissolved in 200 µl of water and the concentration of TNF-alpha was measured. The value was normalized according to the medium volume.

We used an average value of replicate cultures from the same trachea (n = 3) for the analysis of cytokine production.

Northern blot analysis. Northern blot analysis was done as previously described (30, 36). Equal amounts of total RNA (10 µg) extracted from human tracheal epithelial cells, as determined spectrophotometrically, were subjected to electrophoresis in a 1% agarose-formaldehyde gel. The gel was then transferred via capillary action onto a nylon membrane (Hybond-N+, Amersham Life Sciences). The membrane was hybridized with [alpha -32P]dCTP (3,000 Ci/mmol; Amersham)-labeled human ICAM-1 cDNA (1.8-kb XbaI fragment; British Bio-technology, Oxon, UK) with a random-primer labeling kit (Random Primer, Takara). Hybridization with a radiolabeled probe was performed overnight at 42°C. After high-stringency washing was performed (1× saline-sodium citrate and 0.1% sodium dodecyl sulfate, 60°C), autoradiographic detection of the hybridized probe was performed by exposure to Kodak scientific imaging film for 48-72 h at -70°C. Quantification of the autoradiographic bands was accomplished with an image analyzer (Bio Imaging analyzer BAS-2000; Fuji Photo Film).

Effects of bafilomycin A1 on RV infection. Confluent human tracheal epithelial cells were incubated with medium containing bafilomycin A1 (0.1 µM) (26) or with medium alone for 60 min at 37°C. The monolayers were then exposed to RV14 (105 TCID50 units/ml) in MEM containing bafilomycin A1 (0.1 µM) or medium alone for 60 min before being rinsed and the addition of fresh DMEM-Ham's F-12 medium containing 2% USG, 105 U/l of penicillin, 100 mg/l of streptomycin, 50 mg/l of gentamicin, and 2.5 mg/l of amphotericin B supplemented with either bafilomycin A1 (0.1 µM) or the vehicle for bafilomycin A1. Whole volumes of the medium were taken for measurement of the viral content, and the same volume of fresh medium containing either bafilomycin A1 (0.1 µM) or the vehicle for bafilomycin A1 was replaced at various times after infection.

To study the concentration-response effects of bafilomycin A1 on RV infection, concentration-response curves to bafilomycin A1 were obtained with concentrations ranging from 10 pM to 1 µM. To determine the minimum incubation period needed to cause inhibition of RV infection, incubation with bafilomycin A1 (0.1 µM) was also performed for various times ranging from 10 min to 24 h after exposure to RV14 (105 TCID50 units/ml). The cells were then cultured in fresh medium containing the vehicle for bafilomycin A1. To determine whether preincubation with bafilomycin A1 inhibits RV infection, the cells were incubated with bafilomycin A1 (0.1 µM) for either 1 or 24 h and washed. The cells were then exposed to RV14 (105 TCID50 units/ml) containing the vehicle for bafilomycin A1. To determine whether coincubation of RVs with bafilomycin A1 inhibits RV infection, the cells were treated with bafilomycin A1 (0.1 µM) during a period of RV exposure (105 TCID50 units/ml; 60 min). The cells were then cultured in fresh medium containing the vehicle for bafilomycin A1.

We also examined the effects of EIPA and FR-168888, blockers of NHEs (3, 38). Confluent human tracheal epithelial cells were incubated with medium containing either EIPA (10 µM) or FR-168888 (10 µM) from 60 min before RV exposure (105 TCID50 units/ml) to the end of the experiments.

Effects of bafilomycin A1 on the susceptibility to RV infection. To examine whether bafilomycin A1 decreases the susceptibility to RV14 infection, confluent human tracheal epithelial cells were incubated with and without bafilomycin A1 (0.1 µM) for either 1 or 24 h. The epithelial cells were then exposed to serial 10-fold dilutions of RV14 for 1 h at 33°C. The presence of RV14 in the supernatants collected for 1-3 days after infection was determined with the human embryonic fibroblast cell assay described in Detection and titration of viruses to assess whether infection occurred at each dose of RV14 used. This index of susceptibility to infection, defined as the minimum dose of RV14 that could induce infection, was compared with the susceptibility of control cells that were not preincubated with bafilomycin A1 (34).

Detection of RV RNA by RT-PCR. Extraction of RNA from human tracheal epithelial cells cultured in glass tubes was performed as previously described (36), and pellets of RNA were dissolved in water and stored at -80°C before use.

PCR was also performed as previously described (36). Primer pairs for the RV were present at 2 ng/µl. Sequences of the PCR primer pairs used in the present study are described elsewhere (36). The PCR was performed in an automated thermal cycler (MJ Research, Watertown, MA), and 10 µl of the reaction mixture from each sample were removed at 30 cycles. Samples were separated on a 2% agarose gel (FMC BioProducts, Rockland, ME) and stained for 30 min with 1 µg/ml of ethidium bromide. The DNA bands were visualized on a ultraviolet (UV) illuminator and photographed with type 667 positive/negative film (Polaroid, Cambridge, MA).

For the RV, RNA expression in the human tracheal epithelial cells was examined before and 8, 24, 72, and 120 h after RV14 infection in the presence of either bafilomycin A1 (0.1 µM), EIPA (10 µM), FR-168888 (10 µM), or the vehicle for the blockers (control).

Effects of bafilomycin A1 on cytokine production. Because the maximal production of cytokines was obtained on day 3 for IL-1beta , on day 1 for IL-6, on day 1 for IL-8, and on day 3 for TNF-alpha after exposure of human tracheal epithelial cells to RV14 (36), production of IL-1beta , IL-6, IL-8, and TNF-alpha in the culture medium was measured at the maximal production of each cytokine after RV14 infection (105 TCID50 units/ml) in the presence of either bafilomycin A1 (0.1 µM) or the vehicle for bafilomycin A1. The amount of IL-1alpha , IFN-alpha , IFN-beta , and IFN-gamma released from human tracheal epithelial cells into the culture medium was measured before and 1, 3, and 5 days after RV14 infection.

To confirm that increases in cytokine production by RV14 infection were due to the effects of RV14 infection and not to a contaminant present in the viral stock, the ability of UV-inactivated virus to induce cytokine production was also examined. UV inactivation was performed as previously described (16).

As previously demonstrated (36), endogenous IL-1beta is associated with increased production of ICAM-1 after RV14 infection. Furthermore, IL-1beta induces production of various cytokines in airway epithelial cells (4). Therefore, we examined the effects of IL-1beta on the production of IL-6, IL-8, and TNF-alpha and the effects of bafilomycin A1 on the IL-1beta -induced production of these cytokines. To examine the effect of bafilomycin A1 on the IL-1beta -induced production of cytokines in human tracheal epithelial cells, epithelial cells were pretreated with bafilomycin A1 (0.1 µM) for 24 h and then treated with IL-1beta (200 pg/ml) in the presence of bafilomycin A1 (0.1 µM) for 24 or 48 h. The concentration of IL-1beta was matched to a net increase in the culture medium after RV14 infection.

Effects of bafilomycin A1 on ICAM-1 mRNA expression. To study the effects of bafilomycin A1 on ICAM-1 mRNA expression, human tracheal epithelial cells were treated with bafilomycin A1 (0.1 µM) for either 1 or 24 h before RV14 infection (105 TCID50 units/ml). The expression of ICAM-1 mRNA in cells was examined with Northern blot analysis before and 8, 24, 72, and 120 h after RV14 infection. We also studied the effects of EIPA (10 µM) and FR-168888 (10 µM) on the expression of ICAM-1 mRNA 72 h after RV14 infection. We used an average value from replicate cultures of the same trachea (n = 3) for analysis of the intensity of the ICAM-1-to-beta -actin bands.

As previously demonstrated (36), endogenous IL-1beta is associated with increased production of ICAM-1 after RV14 infection. To examine the effect of bafilomycin A1 on the IL-1beta -induced production of ICAM-1 mRNA in human tracheal epithelial cells, epithelial cells were treated with IL-1beta (200 pg/ml) in the presence and absence of bafilomycin A1 (0.1 µM) for 48 h. The concentration of IL-1beta was matched to a net increase in the culture medium after RV14 infection.

Isolation of nuclear extracts. Nuclear extracts were prepared with the methods previously described (27, 47). For the isolation of nuclear extracts, all procedures were performed on ice. Human tracheal epithelial cells were washed with ice-cold PBS, harvested by scraping into PBS, and pelleted in a 1.5-ml microfuge tube at 1,850 g for 5 min. After these procedures were repeated one more time, the pellet was suspended in one packed cell volume of lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] and incubated for 15 min. Membrane lysis was accomplished by adding 25 µl of 10% Nonidet P-40 followed by vigorous agitation. The nuclei were then collected by centrifugation, resuspended in 50 µl of extract buffer (20 mM HEPES, 420 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF), and agitated vigorously at 4°C for 15 min. After removal of debris by centrifugation, the protein concentration of the nuclear extract was determined. The nuclear extracts were then stored at -70°C until used.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assays (EMSAs) were performed as previously described (27, 47). The radiolabeled double-strand oligonucleotide probe for the nuclear factor-kappa B (NF-kappa B) site was prepared by annealing complementary oligonucleotides and by end labeling with [gamma -32P]ATP and T4 polynucleotide kinase. The radiolabeled probe used was composed of the sequence 5'-GATCGAGGGGACTTTCCCTAGC-3' (Stratagene, La Jolla, CA). The labeled probes were purified by CHROMA SPIN + TE-10 (CLONTECH, Palo Alto, CA) and diluted with buffer (10 mM Tris · HCl and 1 mM EDTA) to the desired concentration. Equivalent amounts of nuclear protein were incubated with 2 µg of salmon sperm DNA and 2-5 fmol (20,000 dpm) of the radiolabeled probe for 20 min at room temperature in 20 µl of a buffer containing 10 mM HEPES (pH 7.9), 50 mM KCl, 2 mM MgCl2, 0.25 mM DTT, 0.25 mM PMSF, 0.1 mM EDTA, and 10% glycerol. Resolution was accomplished by electrophoresing 12 µl of reaction solution on 4% nondenaturing polyacrylamide gels in Tris-borate-EDTA buffer (89 mM Tris · HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.0) for 60 min at 150 V at room temperature. Autoradiographic detection of the hybridized probe was performed by exposure to Kodak scientific imaging film for 48-72 h at -70°C.

Supershift EMSA. Supershift assays were used to determine which members of the NF-kappa B family were involved in RV14-induced NF-kappa B-DNA binding. In these studies, EMSAs were performed as described in Electrophoretic mobility shift assay except that rabbit polyclonal antibodies against the NF-kappa B subunit proteins p65, p50, c-Rel, and Rel B (Santa Cruz Biotechnology, Santa Cruz, CA) were included in the 1-h radiolabeled probe-extract binding reaction at 4°C (27, 47). Preimmune serum (Santa Cruz Biotechnology) was used to control for any nonspecific effects of these antisera.

Measurement of intracellular pH. Human tracheal epithelial cells cultured in flasks in DMEM-Ham's F-12 medium containing 2% USG and antibiotics were collected by trypsinization (0.05% trypsin and 0.02% EDTA) and suspended in the Krebs-Ringer phosphate with dextrose solution. Human tracheal epithelial cells (5 × 105 cells) suspended in an Eppendorf tube were incubated at 37°C for 10 min in the Krebs-Ringer phosphate with dextrose solution, which contained 1 µg/ml of 2',7'-bis-(carboxyl)-5-(and-6)-carboxylfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR). After BCECF loading, the cells were centrifuged at 200 g for 5 min at 37°C and resuspended in a thermostated stirred cuvette, and fluorescence was measured with excitation at 490 nm and emission at 525 nm with 5-nm slits in a fluorescence spectrophotometer (Hitachi F-2000, Tokyo, Japan) as previously described (9). Intracellular pH (pHi) was calibrated with nigericin and K+ as previously described (37). We found in the preliminary experiments that sodium propionate induced a transient cytosolic acidification of the epithelial cells followed by a prolonged alkalinization. Therefore, we examined the effect of bafilomycin A1 (0.1 µM), EIPA (10 µM), and FR-168888 (10 µM) on the recovery of pHi. The recovery of pHi after the addition of sodium propionate is expressed as the initial recovery rate of pHi. The initial recovery rate of pHi was determined by measuring the slope of the line tangent to the initial upstroke of the curve and calculating the change in pHi for 1 min (20).

Measurement of changes in acidic endosome distribution. Human tracheal epithelial cells were cultured on glass-bottom microwell dishes (Mat Tek, Ashland, MA). To examine the distribution of acidic endosomes in human tracheal epithelial cells, we stained the endosomes in the cells with a dye, LysoSensor Green DND-189 (Molecular Probes), according to the manufacturer's instructions (13). LysoSensor Green DND-189 detects pH values in the 4.5-6.0 range (13). The cells were washed twice with PBS at room temperature, incubated for 15 min in PBS containing 1 µM LysoSensor Green DND-189 and 5 mM glucose, and observed under confocal fluorescent microscopy (Leica DMRXE) with a ×40 objective. Images of acidic endosomes and differential interference microscopy of human tracheal epithelial cells were recorded on a Leica TCS NT system every 6 s for 400 s and used for analysis.

Preparation of the drugs. Bafilomycin A1, EIPA, and FR-168888 were dissolved in 100% dimethyl sulfoxide to give stock solutions of 0.1 mM bafilomycin A1 and 10 mM EIPA and FR168888 and stored at 4°C. The stock solution was diluted in the culture medium as necessary for each experiment.

Statistical analysis. The results are expressed as means ± SE; n is the number of donors from whom cultured epithelial cells were used. Statistical analysis was performed with two-way repeated-measures ANOVA. Subsequent post hoc analysis was made with Bonferroni's method. For all analysis, values of P < 0.05 were assumed to be significant.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of bafilomycin A1 on RV infection in human tracheal epithelial cells. Exposing confluent tracheal epithelial cell monolayers to RV14 (105 TCID50 units/ml) consistently led to infection. The collection of culture medium at differing times after viral exposure revealed no detectable virus 1 h after infection. RV14 was detectable in the culture medium 6 h after infection, and the viral content progressively increased between 6 and 24 h after infection (Fig. 1A). Evidence of continuous viral production was obtained by demonstrating that the viral titers of supernatants collected for 1-3, 3-5, and 5-7 days after infection each contained significant levels of RV14 (Fig. 1B). Treatment of the cells with bafilomycin A1 (0.1 µM) significantly decreased the viral titers in supernatants from 12 h after infection (Fig. 1).


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Fig. 1.   A: viral titer in supernatants of human tracheal epithelial cells collected at sequential times during the 1st 24 h after exposure to 105 units of 50% tissue culture infectious dose (TCID50)/ml of rhinovirus (RV) 14 in the presence () and absence (open circle ) of bafilomycin A1 (0.1 µM). B: viral titer in supernatants collected for indicated days after exposure to 105 TCID50 units/ml of RV14 in the presence (solid bars) and absence (open bars) of bafilomycin A1 (0.1 µM). The cells were exposed to bafilomycin A1 or vehicle for bafilomycin A1 from 60 min before RV14 infection to day 7 after infection. Results are means ± SE of separate experiments with cells from 7 different donors. ** Significant difference from viral infection alone, P < 0.01.

The inhibitory effects of bafilomycin A1 on RV14 infection were concentration dependent, and the maximum effect was obtained at 0.1 µM (Fig. 2A). Likewise, the inhibitory effects of bafilomycin A1 (0.1 µM) on RV14 infection were dependent on the incubation period after exposure to RV14, and the minimum incubation time to cause inhibition of RV14 infection was obtained at 30 min (Fig. 2B). When the cells were pretreated with bafilomycin A1 (0.1 µM) for 24 h and bafilomycin A1 was then removed, the viral titers of RV14 in the supernatants became the same as those in the cells without bafilomycin A1 pretreatment 5 days after infection (Fig. 2C). Furthermore, when the cells were treated with bafilomycin A1 for 1 h during the exposure to RV14, the viral titers of RV14 in the supernatants became the same as those in the cells without bafilomycin A1 treatment 3 days after infection (Fig. 2C). However, bafilomycin A1 (0.1 µM) failed to inhibit the viral titers of RV14 in the supernatants when the cells were pretreated with bafilomycin A1 for 1 h before exposure to RV14 (4.1 ± 0.2 TCID50 units/ml in the presence of bafilomycin A1 vs. 4.2 ± 0.2 TCID50 units/ml in the absence of bafilomycin A1 1 day after infection; P > 0.20; n = 7).


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Fig. 2.   A: concentration-response effect of bafilomycin A1 on RV14 infection in human tracheal epithelial cells. Viral titers were determined in supernatants collected for 1-3 days after RV14 infection (105 TCID50 units/ml). The cells were exposed to bafilomycin A1 from 60 min before RV14 infection to day 3 after infection. Results are means ± SE of separate experiments with cells from 7 different donors. Significant difference from corresponding control values: * P < 0.05; ** P < 0.01. B: effect of incubation time of bafilomycin A1 (0.1 µM) on viral titer in supernatants collected for 1-3 days after exposure to 105 TCID50 units/ml of RV14. The cells were exposed to bafilomycin A1 for various times after RV14 infection. The cells were then cultured in the medium containing the vehicle for bafilomycin A1. Results are means ± SE of separate experiments with cells from 7 different donors. Significant difference from control value (day 0): * P < 0.05; ** P < 0.01; *** P < 0.001. C: viral titer in supernatants 1, 3, 5, and 7 days after exposure to 105 TCID50 units/ml of RV14. Cells were exposed to bafilomycin A1 (0.1 µM) before RV14 infection for 24 h () or to bafilomycin A1 (0.1 µM) during RV14 infection for 1 h (triangle ). The cells were then cultured in the medium containing the vehicle for bafilomycin A1. open circle , Control cells. Results are means ± SE of separate experiments with cells from 7 different donors. Significant difference from corresponding control value: * P < 0.05; **P < 0.01.

The viral titers of the cell lysates also contained significant levels of RV14. The viral titers of cell-associated RV14 were 0.0 ± 0.0 log TCID50 units at 1 h, 0.1 ± 0.1 log TCID50 units at 6 h, 0.8 ± 0.1 log TCID50 units at 12 h, 2.2 ± 0.2 log TCID50 units at 24 h, 2.4 ± 0.3 log TCID50 units at 3 days, 2.2 ± 0.2 log TCID50 units at 5 days, and 1.8 ± 0.2 log TCID50 units at 7 days (n = 7 each). In the cell lysates, the viral titer levels also increased significantly with time (P < 0.05 in each case by ANOVA). Bafilomycin A1 (0.1 µM) significantly decreased the cell-associated viral titers of RV14. The viral titers of cell-associated RV14 in bafilomycin A1-treated human tracheal epithelial cells were 0.0 ± 0.0 log TCID50 units at 1 h, 0.0 ± 0.0 log TCID50 units at 6 h, 0.0 ± 0.0 log TCID50 units at 12 h (P < 0.01), 0.7 ± 0.1 log TCID50 units at 24 h (P < 0.05), 1.2 ± 0.2 log TCID50 units at 3 days (P < 0.05), 1.1 ± 0.2 log TCID50 units at 5 days (P < 0.05), and 1.0 ± 0.1 log TCID50 units at 7 days (P < 0.05; n = 7 each).

Human tracheal cell viability as assessed by the exclusion of trypan blue was consistently >96% in the RV-infected and bafilomycin A1 (0.1 µM; 5 days)-treated cultures. Likewise, RV14 infection did not alter the amount of lactate dehydrogenase in the supernatants (30 ± 3 IU/l before vs. 31 ± 3 IU/l 5 days after infection; P > 0.50; n = 7). Both RV14 infection (105 TCID50 units/ml; 5 days) and bafilomycin A1 treatment (0.1 µM; 5 days) also had no effect on cell number and total protein content when RV14 infection and bafilomycin A1 treatment were performed after the cells had formed confluent cell sheets in the tubes. Cell counts after RV14 infection (1.8 × 106 ± 0.2 × 106 cells; P > 0.50; n = 7) and with bafilomycin A1 treatment (1.8 × 106 ± 0.1 × 106 cells; P > 0.50; n = 7) were not significantly different from the control value (1.8 × 106 ± 0.1 × 106 cells; n = 7). Total protein contents after RV14 infection (1,012 ± 107 µg/106 cells; P > 0.50; n = 7) and with bafilomycin A1 treatment (1,023 ± 155 µg/106 cells; P > 0.50; n = 7) were not significantly different from the control value (1,015 ± 124 µg/106 cells; n = 7). Exposure to bafilomycin A1 for 5 days did not change the baseline electrical resistance and potential difference of the cultured human tracheal epithelial cells. The baseline electrical resistance 5 days after exposure to bafilomycin A1 (0.1 µM) was 141 ± 17 Omega  · cm2 (P > 0.20) compared with 152 ± 25 Omega  · cm2 in control cells (n = 5). The baseline potential difference 5 days after exposure to bafilomycin A1 (0.1 µM) was 9.2 ± 0.3 mV compared with 10.3 ± 0.9 mV in control cells (P > 0.20; n = 5). Likewise, EIPA (10 µM) and FR-168888 (10 µM) did not change the baseline electrical resistance. The baseline electrical resistance 5 days after exposure to EIPA (10 µM) and FR-168888 (10 µM) was 148 ± 22 (P > 0.50) and 154 ± 22 Omega  · cm2 (P > 0.50), respectively (n = 5). EIPA (10 µM) slightly decreased the potential difference, but FR-168888 (10 µM) was without effect on the potential difference. The potential difference 5 days after exposure to EIPA (10 µM) and FR-168888 (10 µM) was 7.4 ± 1.1 (P < 0.05) and 9.9 ± 1.0 mV (P > 0.50), respectively (n = 5).

Viral titers during the 1-3 days after RV14 infection in supernatants of the cells treated with EIPA (10 µM; 2.9 ± 0.2 log TCID50 units/ml; P < 0.05; n = 7) and FR-168888 (10 µM; 3.1 ± 0.2 log TCID50 units/ml; P < 0.05; n = 7) significantly decreased compared with that in control cells (4.2 ± 0.3 log TCID50 units/ml; n = 7), although the inhibitory effects of EIPA and FR-168888 were smaller than those of bafilomycin A1 (0.1 µM; 1.1 ± 0.2 log TCID50 units/ml; P < 0.05; n = 7).

Effects of bafilomycin A1 on the susceptibility to RV infection. Pretreatment of human tracheal epithelial cells with bafilomycin A1 (0.1 µM) for 24 h decreased the susceptibility of cells to RV14 infection, increasing by 10-fold the minimum dose of virus necessary to cause infection (3.7 ± 0.2 log TCID50 units/ml; P < 0.01; n = 7) compared with that in medium alone (2.1 ± 0.2 log TCID50 units/ml; n = 7). In contrast, 1 h of pretreatment with bafilomycin A1 did not increase the susceptibility to RV14 infection (data not shown).

Detection of viral RNA by PCR. Further evidence of the effects of bafilomycin A1, EIPA, and FR-168888 on RV14 infection in human tracheal epithelial cells was provided by PCR analysis. In each of three experiments, RNA extracted from control uninfected cells did not produce any detectable PCR product at 381 bp (0 h). A product band was observed in RNA extracted from cells 24 h after infection followed by increases in viral RNA 72 and 120 h after infection (Fig. 3). The time course of the viral titers in the supernatants as detected by the cytopathic effects of viruses on human embryonic fibroblast cells was similar to that of the intensity of the RV RNA product extracted from infected cells detected by PCR analysis. Both bafilomycin A1 (0.1 µM) and EIPA (10 µM) reduced the intensity of the RV RNA product extracted from infected cells (Fig. 3). Likewise, FR-168888 (10 µM) reduced the intensity of the RV RNA products extracted from infected cells (data not shown).


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Fig. 3.   Time course of replication of RV RNA in lysates of human tracheal epithelial cells after RV14 infection as detected by RT-PCR in the presence of either 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 10 µM), bafilomycin A1 (BA1; 0.1 µM), or water as a vehicle for blockers (control). The cells were exposed to blockers or vehicle for blockers from 60 min before RV14 infection to 120 h after infection. A positive signal is demonstrated by a band at 381 bp. beta -Actin was used as a housekeeping gene. M, Phi X174-HincII fragment molecular weight marker. Data are representative of 3 different experiments.

Effects of bafilomycin A1 on cytokine production. Because both viral infection and bafilomycin A1 treatment did not alter cell number, all cytokine values are reported in picograms per milliliter of supernatant. Figure 4 shows that secretion of IL-1beta , IL-6, IL-8, and TNF-alpha all increased in response to RV14. However, increases in cytokine production in cells with UV-inactivated RV14 infection were similar to those in cells with a sham infection. Bafilomycin A1 decreased RV14 infection-induced increases in the production of IL-1beta , IL-6, IL-8, and TNF-alpha as well as the basal secretion of IL-1beta and IL-8 (Fig. 4). The inhibitory effect of bafilomycin A1 on the baseline production of IL-8 was smaller (76% of control value) than that of IL-1beta (57% of control value), although bafilomycin A1 decreased the production of IL-1beta and IL-8 after RV14 infection to the baseline levels. In contrast, RV14 infection did not alter IL-1alpha production (13 ± 2 pg/ml 3 days after RV14 infection vs. 12 ± 2 pg/ml 3 days after sham infection; P > 0.20; n = 7). Of the cytokines measured, IFN-alpha , IFN-beta , and IFN-gamma were under the limit of detection of the assay in the supernatants from cells with RV14 and sham infections throughout the experiments.


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Fig. 4.   Effect of BA1 (0.1 µM) on the release of either interleukin (IL)-1beta (A), IL-6 (B), IL-8 (C), or tumor necrosis factor (TNF)-alpha (D) into culture supernatants of human tracheal epithelial cells after RV14, ultraviolet (UV)-inactivated RV14, or sham (control) infection. The effects of BA1 were examined at maximal production of each cytokine after RV14 infection. The cells were exposed to BA1 or vehicle for BA1 from 24 h before RV14 infection to the end of the experiments. Results are means ± SE of separate experiments with cells from 7 different donors. Significant difference from corresponding control value: * P < 0.05; ** P < 0.01; *** P < 0.001. Significant difference from RV14 infection alone: dagger  P < 0.05; dagger dagger P < 0.01.

IL-1beta (200 pg/ml) increased the production of IL-6 (42 ± 3 pg/ml with IL-1beta vs. 27 ± 2 pg/ml in control cells; P < 0.05; n = 7), IL-8 (671 ± 35 pg/ml with IL-1beta vs. 592 ± 37 pg/ml in control cells; P < 0.05; n = 7), and TNF-alpha (3.2 ± 0.2 pg/ml with IL-1beta vs. 0 ± 0 pg/ml in control cells; P < 0.05; n = 7). However, bafilomycin A1 failed to inhibit the production of these cytokines induced by IL-1beta (41 ± 3 pg/ml of IL-6; 659 ± 36 pg/ml of IL-8; 2.9 ± 0.2 pg/ml of TNF-alpha ; P > 0.50; n = 7).

Effects of bafilomycin A1 on ICAM-1 mRNA expression. Exposure of the cells to RV14 caused increases in ICAM-1 mRNA in the absence of bafilomycin A1 (Fig. 5A). Human tracheal epithelial cells 3 days after RV14 infection were shown to overexpress ICAM-1 mRNA twofold compared with that 3 days after sham exposure (control; Fig. 5B). Bafilomycin A1 (0.1 µM) inhibited increases in ICAM-1 mRNA induced by RV14 infection without effects on beta -actin mRNA (Fig. 5A) and significantly decreased ICAM-1 mRNA as well as baseline ICAM-1 mRNA expression 3 days after RV14 infection (Fig. 5B). However, preincubation with bafilomycin A1 (0.1 µM) for 1 h did not change ICAM-1 mRNA expression in the human tracheal epithelial cells (0.21 ± 0.02 scan units with bafilomycin A1 vs. 0.21 ± 0.02 scan units in control cells; P > 0.50; n = 7). IL-1beta (200 pg/ml) increased ICAM-1 mRNA (0.35 ± 0.04 scan units vs. 0.20 ± 0.02 scan units in control cells; P < 0.01; n = 7). In contrast, bafilomycin A1 failed to inhibit IL-1beta -induced-ICAM-1 mRNA expression (0.34 ± 0.03 scan units; P > 0.50; n = 7).


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Fig. 5.   A: Northern blot analysis for intercellular adhesion molecule (ICAM)-1 mRNA levels in human tracheal epithelial cells before (0) and 8, 24, 72, and 120 h after RV14 infection in the presence (+) and absence (-) of BA1 (0.1 µM). beta -Actin was used as a housekeeping gene. B: effects of BA1 (0.1 µM) on the expression of ICAM-1 mRNA in human tracheal epithelial cells 3 days after RV14 or sham (control) infection. The cells were exposed to BA1 or vehicle for BA1 from 24 h before RV14 infection to the end of the experiments. ICAM-1 mRNA was normalized to constitutive expression of beta -actin mRNA. Results are means ± SE of separate experiments with cells from 7 different donors. Significant difference from corresponding control value: * P < 0.05; ** P < 0.01. ++ Significant difference from RV14 infection alone, P < 0.01.

EIPA (10 µM for 72 h) and FR-168888 (10 µM for 72 h) inhibited RV14 infection-induced increases in mRNA expression of ICAM-1 (0.35 ± 0.03 scan units with EIPA and 0.37 ± 0.03 scan units with FR-168888; P < 0.05; n = 7) compared with that in control cells (0.46 ± 0.03 scan units; n = 7), although the inhibitory effects of EIPA and FR-168888 were smaller than those of bafilomycin A1 (0.1 µM; 0.24 ± 0.03 scan units; P < 0.01; n = 7).

NF-kappa B DNA binding activity in human tracheal epithelial cells. Nuclear extracts from the human tracheal epithelial cells with RV14 or sham infection contained activated NF-kappa B as demonstrated by the presence of a complex consisting of a protein bound to a DNA fragment carrying NF-kappa B (Fig. 6). The baseline intensity of NF-kappa B binding activity was constant, and increased activation of NF-kappa B was present in cells from 30 min after RV14 infection (Fig. 6). Bafilomycin A1 (0.1 µM) reduced the increased activation of NF-kappa B by RV14 infection as well as the baseline intensity of NF-kappa B binding activity (Fig. 6). Specificity of the NF-kappa B binding was confirmed by supershift EMSA in which antibodies to the p50 or p65 subunit of NF-kappa B ablated NF-kappa B bands (Fig. 7). Supershifting of the NF-kappa B band with the antibody to the p50 or p65 subunit of NF-kappa B was constantly observed at any time of cell culture. However, supershifting of the NF-kappa B band was not observed with the antibody to either p52, c-Rel, Rel B, or preimmune antiserum (Fig. 7).


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Fig. 6.   Electrophoretic mobility shift assay demonstrating the effects of BA1 (0.1 µM) on the increases in nuclear factor (NF)-kappa B DNA binding activity of human tracheal epithelial cells before (0) and 30, 60, and 120 min after RV14 infection. The cells were exposed to BA1 or vehicle for BA1 from 24 h before RV14 infection to the end of the experiments. Arrows, RV14-induced NF-kappa B DNA binding activity.



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Fig. 7.   Identification of RV14-induced NF-kappa B bands with supershift electrophoretic mobility shift assay (EMSA). EMSAs were performed in the presence and absence of antisera against NF-kappa B family proteins, and antisera against p50, p65, p52, c-Rel, and Rel B were compared with preimmune serum. Arrows, RV14-induced NF-kappa B DNA binding activity. Arrowhead, supershifted bands caused by antisera against p50 and p65.

Effects of blockers of V-ATPases and NHEs on pHi. Sodium propionate (10 µM) induced a transient cytosolic acidification of the epithelial cells followed by a prolonged alkalinization (Fig. 8A). Bafilomycin A1, EIPA, and FR-168888 each inhibited the alkalinization of the epithelial cells after the addition of sodium propionate without changes in the baseline pHi (Fig. 8, B-D). The potency order of the inhibitory effects on alkalinization after sodium propionate was similar among these three blockers (Fig. 8E). The inhibitory effects of bafilomycin A1 was concentration dependent, and the maximum effect was obtained at 0.1 µM (Fig. 8F).


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Fig. 8.   Typical time courses of intracellular pH (pHi) in response to sodium propionate (10 µM) in the absence (A) and presence of BA1 (0.1µM; B), EIPA (10 µM; C), or FR-168888 (10 µM; D). Arrows, time of administration of sodium propionate. E: effects of BA1 (0.1 µM), EIPA (10 µM), and FR-168888 (10 µM) on changes in pHi (dpHi/dt) after acidification by sodium propionate (10 µM). Results are means ± SE of separate experiments with cells from 7 different donors. *** Significant difference from corresponding control value (control), P < 0.001. F: concentration-response effects of BA1 on dpHi/dt after acidification by sodium propionate (10 µM). The cells were exposed to blockers or vehicle for blockers from 5 min before the addition of sodium propionate to the end of the experiments. Results are means ± SE of separate experiments with cells from 7 different donors. Significant difference from corresponding control value: * P < 0.05; ** P < 0.01; *** P < 0.001.

Effects of bafilomycin A1 on the acidification of endosomes. Acidic endosomes in human tracheal epithelial cells were stained green with a dye, LysoSensor Green DND-189. Figure 9 shows the time-course changes in the distribution and the fluorescence intensity of acidic endosomes with green fluorescence in the human tracheal epithelial cells. Green fluorescence was observed in a granular pattern in the cytoplasm. The distribution and intensity of the green fluorescence were constant for at least 300 s in the epithelial cells cultured in PBS alone (Fig. 9, A and C). Bafilomycin A1 (0.1 µM) significantly decreased the number and fluorescence intensity of the acidic endosomes with green fluorescence in the human tracheal epithelial cells at 50 s, and the fluorescence intensity in the cells was markedly reduced 300 s after the addition of bafilomycin A1 (Fig. 9, B and C). Likewise, either EIPA (10 µM) or FR-168888 (10 µM) decreased the green fluorescence intensity of acidic endosomes in the human tracheal epithelial cells (Fig. 9C). The potency order of the inhibitory effects based on fluorescence intensity was bafilomycin A1 > EIPA > FR-168888 (Fig. 9C).


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Fig. 9.   Changes in the distribution of acidic endosomes with green fluorescence in human tracheal epithelial cells 100 s before (-100s) and 300 s after treatment with BA1 (0.1 µM; B) or vehicle for BA1 (control; A). C: time-course changes in the intensity of green fluorescence from acidic endosomes in human tracheal epithelial cells after treatment with either BA1 (0.1 µM; ), EIPA (10 µM; ), FR-168888 (10 µM; black-triangle), or vehicle for BA1 (control; open circle ). Inhibitors were administrated at time 0. Acidic endosomes in human tracheal epithelial cells were stained green with a dye, LysoSensor Green DND-189. Data are representative of 3 different experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have shown that a specific inhibitor of V-ATPase, bafilomycin A1, reduced the viral titers of a major RV subgroup, RV14, in the supernatants and reduced the cell-associated viral titers and viral RNA in cultured human tracheal epithelial cells. The inhibitory effects on the viral titers of RV14 were observed by incubation with bafilomycin A1 either during or after RV14 infection. Furthermore, the inhibitory effects of bafilomycin A1 on RV14 infection were obtained within 30 min after RV14 infection and at a concentration of bafilomycin A1 > 1 pM. These findings are consistent with previous reports for HeLa cells and MDCK cells (26, 28). Furthermore, bafilomycin A1 decreased the number of acidic endosomes distributed in a granular pattern in the cytoplasm of human tracheal epithelial cells. Therefore, bafilomycin A1 may increase the pH of endosomes and inhibit RV14 infection via the inhibitory effects on the entry of RV14 RNA from acidic endosomes to the cytoplasm in the airway epithelial cells as reported in HeLa cells (2, 28).

Furthermore, pretreatment with bafilomycin A1 for 24 h also inhibited the viral titers of RV14 and ICAM-1 mRNA expression and reduced the susceptibility to RV14 in the present study. The viral titers of the supernatants recovered 5 days after removal of bafilomycin A1 when the cells were pretreated with bafilomycin A1 for 24 h. These findings suggest that bafilomycin A1 may also inhibit infection of RV14 by reducing its receptor, ICAM-1 (12), in airway epithelial cells. In contrast, 1 h of bafilomycin A1 pretreatment failed to inhibit RV14 infection, probably due to washout of bafilomycin A1 during 1 h of RV14 infection. Furthermore, 1 h of bafilomycin A1 pretreatment did not increase the susceptibility to RV14 infection, suggesting that 1 h of bafilomycin A1 pretreatment might not reduce the production of ICAM-1, the receptor for RV14 (12), in the human tracheal epithelial cells. Brefeldin A, another macrolide antibiotic, is reported to block protein glycosylation and RNA replication of vesicular stomatitis virus (17). Further study is needed to examine the precise mechanisms.

Recent reports (2, 3, 7, 15, 28, 29) revealed the mechanisms of RV entry into the cells. RV14 forms RV-soluble ICAM-1 complexes, and these complexes can release viral RNA (7, 15). Furthermore, RV14 releases RNA after exposure to acidic pH (7), and infection of RV14 in HeLa cells is inhibited by bafilomycin A1 (28). Therefore, the entry of RV14 RNA into the cells is suggested to be mediated by the destabilization after binding to ICAM-1, by endosomal acidification, or both (7).

pHi is suggested to be regulated by ion transport mechanisms across various antiporters and exchangers such as Na+/H+ antiporters (NHEs), H+-ATPases (V-ATPase), and Cl--HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers in the cell membrane and vacuoles (19, 35). Endosomal pH is also suggested to be regulated by ion transport across the endosomal V-ATPase and NHEs (10, 21, 23, 46). In the present study, EIPA and FR-168888, inhibitors of NHEs, and bafilomycin A1, an inhibitor of V-ATPase, reduced pHi and increased endosomal pH. These findings are consistent with previous reports (19, 21, 39). Therefore, both NHEs and V-ATPase may be distributed in both the cell membrane and the endosomes in human tracheal epithelial cells and may regulate pHi and endosomal pH. Furthermore, the potency order of the inhibitory effects of bafilomycin A1, EIPA, and FR-168888 on the RV titers was correlated with that of the inhibitory effects on the fluorescence intensity of the acidic endosomes. RV14 may therefore enter human tracheal epithelial cells across the acidic endosome caused by NHEs and V-ATPase.

Bafilomycin A1 inhibited not only RV14 infection but also the production of cytokines when the cells were treated with bafilomycin A1 before and after RV14 infection in the present study. In contrast, 1 h of pretreatment with bafilomycin A1 failed to inhibit the production of cytokines. Short periods of bafilomycin A1 pretreatment might not be sufficient for the inhibition of cytokine production in the subsequent epithelial cell culture for 24 or 48 h. The role of V-ATPases on the production of cytokines in the airway epithelial cells is uncertain. However, V-ATPases have a number of functions such as providing the acidic environment associated with the dissociation of internalized ligand-receptor complexes within endosomes and activating the secreted lysosomal enzymes and degradation of the bone matrix (10). Furthermore, acidic pH provided by V-ATPases appears to play a role in the delivery of soluble proteins in the cells (42). Therefore, it is possible that bafilomycin A1 may affect the production of cytokines by, in part, inhibiting the function of intracellular organelles such as Golgi apparatus (10, 22).

The inhibitory effect of bafilomycin A1 on the baseline production of IL-8 was smaller (76% of control value) than that of IL-1beta (57% of control value), although bafilomycin A1 decreased the production of IL-1beta and IL-8 after RV14 infection to the baseline levels. The precise reason is uncertain. However, the inhibitory effects of bafilomycin A1 on cytokine production may differ among cytokine species because the amount of cytokine production differed among cytokine species in the same cell sheets in the present study.

Nuclear extracts from the human tracheal epithelial cells cultured in medium alone contained activated NF-kappa B, and bafilomycin A1 reduced the activated NF-kappa B before RV14 infection in the present study. Furthermore, increased activation of NF-kappa B was present in the cells from 0.5 h after RV14 infection. Bafilomycin A1 also inhibited the increased activation of NF-kappa B after RV infection. The time course of NF-kappa B activation is consistent with previous reports (27, 47) in airway epithelial cells caused by RV14 or RV16 infection. RV infection increases the production of proinflammatory cytokines and ICAM-1 in airway epithelial cells (27, 34, 36, 47). The expression of genes for many cytokines and ICAM-1 induced by RV infection are suggested to be mediated by the activation of NF-kappa B (27, 47). Furthermore, inhibition of NF-kappa B activation by bafilomycin A1 in human tracheal epithelial cells is consistent with a previous report (45) in B lymphocytes and monocyte-like cell lines. Therefore, bafilomycin A1 might reduce production of cytokines and ICAM-1 mRNA before and after RV14 infection via, in part, inhibiting the activation of NF-kappa B in epithelial cells.

In the present study, we observed that RV14 infection increased production of IL-1beta , IL-6, IL-8, and TNF-alpha in cultured human tracheal epithelial cells. Of these, IL-1beta is suggested to upregulate ICAM-1 expression in cultured human tracheal epithelial cells (36). IL-1beta is a potent inflammatory cytokine that induces growth and differentiation of T and B lymphocytes, other cytokine production, prostaglandin E2 synthesis, and degranulation from neutrophils (1). IL-1beta also causes increases in ICAM-1 expression on both epithelial and vascular endothelial cells (8, 38). The epithelial cells in the human airway express ICAM-1 on their surface, which is the site of attachment for 90% of the ~100 RV serotypes (12, 32). ICAM-1 interacts physiologically with leukocyte function-associated antigen-1, expressed on leukocytes, and thus plays a vital role in the recruitment and migration of immune effector cells to sites of local inflammation. Recent studies (27, 31, 36) have shown that RV infection upregulates ICAM-1 expression on the airway epithelial cells, an effect that would facilitate viral cell attachment and entry. Increases in ICAM-1 mRNA expression on human tracheal epithelial cells induced by RV14 infection in the present study are in accord with those in the previous studies.

In summary, the specific V-ATPase blocker bafilomycin A1 may inhibit RV14 infection in human airway epithelial cells by blocking RNA entry into the endosomes. Furthermore, bafilomycin A1 may decrease the susceptibility of cultured human tracheal epithelial cells to RV14 infection, probably via inhibition of ICAM-1, and reduce inflammatory cytokine production by human tracheal epithelial cells in RV14 infection. Bafilomycin A1 failed to inhibit IL-1beta -induced increases in the production of IL-6, IL-8, TNF-alpha , and mRNA of ICAM-1. In contrast, bafilomycin A1 inhibited RV14 infection-induced production of cytokines and ICAM-1, suggesting that the effects of bafilomycin A1 were specific for the RV14 infection-induced cell activation. These findings suggest that bafilomycin A1 may modulate inflammatory responses in airway epithelial cells after RV14 infection.


    ACKNOWLEDGEMENTS

We thank Grant Crittenden for the English corrections and Akira Ohmi, Michiko Okamoto, Minako Tada, and Fusako Chiba for technical assistance.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Sasaki, Dept. of Geriatric and Respiratory Medicine, Tohoku Univ. School of Medicine, Seiryo-machi, Aoba-ku 1-1, Sendai 980-8574, Japan (E-mail: dept{at}geriat.med.tohoku.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 April 2000; accepted in final form 26 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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