Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1beta

Masanori Terajima, Mutsuo Yamaya, Kiyohisa Sekizawa, Shoji Okinaga, Tomoko Suzuki, Norihiro Yamada, Katsutoshi Nakayama, Takashi Ohrui, Takeko Oshima, Yoshio Numazaki, and Hidetada Sasaki

Department of Geriatric Medicine, Tohoku University School of Medicine, Sendai 980; and Virus Center, Clinical Research Division, Sendai National Hospital, Sendai 983, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Exacerbations of asthma are often associated with respiratory infection caused by rhinoviruses. To study the effects of rhinovirus infection on respiratory epithelium, a primary target for respiratory viruses, human rhinovirus (HRV)-2 and HRV-14 were infected to primary cultures of human tracheal epithelial cells. Viral infection was confirmed by showing that viral titers of supernatants and lysates from infected cells increased with time and by polymerase chain reaction. HRV-2 and HRV-14 infections upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, the major rhinovirus receptor, on epithelial cells, and they increased the production of interleukin (IL)-1beta , IL-6, IL-8, and tumor necrosis factor (TNF)-alpha in supernatants. Antibodies to ICAM-1 inhibited HRV-14 infection of epithelial cells and decreased the production of cytokines after HRV-14 infection, but they did not alter HRV-2 infection-induced production of cytokines. IL-1beta upregulated ICAM-1 mRNA expression and increased susceptibility to HRV-14 infection, whereas other cytokines failed to alter ICAM-1 mRNA expression. Furthermore, a neutralizing antibody to IL-1beta significantly decreased viral titers of supernatants and ICAM-1 mRNA expression after HRV-14 infection, but a neutralizing antibody to TNF-alpha was without effect. Immunohistochemical studies revealed that both HRV-14 infection and IL-1beta increased ICAM-1 expression on cultured epithelial cells. These findings imply that HRV-14 infection upregulated ICAM-1 expression on epithelial cells through increased production of IL-1beta , thereby increasing susceptibility to infection. These events may be important for amplification of airway inflammation after viral infection in asthma.

asthma; common cold; airway inflammation; interleukin-1beta ; intercellular adhesion molecule-1; polymerase chain reaction

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PERSPECTIVE STUDIES have indicated that asthma attacks are associated with a viral infection in as many as 20-50% of the cases (17). Rhinoviruses cause the majority of common colds, which often provoke wheezing in patients with asthma (18), and they have also been associated with exacerbations of chronic bronchitis (11). Although several mechanisms have been proposed, it is still uncertain how viral respiratory infections cause an attack of wheezing in patients with asthma.

Respiratory tract epithelium is the primary target for respiratory viruses. Respiratory syncytial viruses cause an increase in interleukin (IL)-8 mRNA expression of the nasal epithelium (4) and the production of IL-6, IL-8, and granulocyte macrophage colony-stimulating factor (GM-CSF) from a human bronchial epithelial cell line (BEAS-2B; see Ref. 19). Likewise, human rhinovirus (HRV) infection induces production of IL-6, IL-8, and GM-CSF from BEAS-2B cells (27). These cytokines are known to mediate a wide variety of proinflammatory and immunoregulatory effects (1) and to play a role in the pathogenesis of airway inflammation in asthma. Subauste et al. (27) demonstrated that preexposure of BEAS-2B cells to tumor necrosis factor-alpha (TNF-alpha ) increased susceptibility to HRV-14 infection. However, HRV-14 infection itself failed to increase susceptibility to infection and intercellular adhesion molecule-1 (ICAM-1) expression, a major surface receptor for rhinoviruses (7). We speculate that a lack of increased susceptibility to infection may be due to the use of the BEAS-2B cell line in the study by Subauste et al. (27). The BEAS-2B cell was derived from normal human epithelial cells by transfection with an adenovirus 12-SV40 hybrid virus (25). Although BEAS-2B cells share many properties in common with normal epithelial cells (27), transformed cell lines may lose certain differentiated functions (8), resulting in an altered response of respiratory epithelial cells to rhinovirus infection.

We therefore investigated whether the primary cultures of the human tracheal epithelial cells (34) can be infected with rhinoviruses. We also examined whether rhinovirus infection upregulates ICAM-1 expression on the cells and increases susceptibility to infection.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Media components. Reagents for cell culture media were obtained as follows: Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, fetal calf serum (FCS), and gamma -globulin-free calf serum (GGFCS) were from GIBCO-BRL Life Technologies, Palo Alto, CA; trypsin, EDTA, dithiothreitol, Sigma type XIV protease, human placental collagen, penicillin, streptomycin, gentamicin, and amphotericin B were from Sigma Chemical, St. Louis, MO; and Ultroser G serum substitute (USG) was from BioSepra, Marlborough, MA.

Human embryonic fibroblast cell culture. Human embryonic fibroblast cells were cultured in the Roux type bottle (Iwaki Garasu, Chiba, Japan) sealed with a rubber plug in MEM containing 10% FCS supplemented with 5 × 104 U/l penicillin and 50 mg/l streptomycin (20). Confluency was achieved at 7 days, at which time the cells were collected by trypsinization (0.05% trypsin, 0.02% EDTA). Cells (1.5 × 105 cells/ml) suspended in MEM containing 10% FCS were then plated in glass tubes (15 × 105 mm; Iwaki Garasu), sealed with rubber plugs, and cultured at 37°C.

Human tracheal epithelial cell culture. Tracheas for cell culture were obtained 3-6 h after death from 51 patients (mean age, 64 ± 4 yr; 22 female, 29 male) under a protocol passed by the Tohoku University Ethics Committee. Twenty-four of the patients were smokers. None had a respiratory illness, and they died of acute myocardial infarction (n = 13), congestive heart failure (n = 3), malignant tumor other than lung cancer (n = 14), rupture of aortic aneurysm (n = 4), liver cirrhosis (n = 3), renal failure (n = 3), leukemia (n = 3), malignant lymphoma (n = 1), cerebral bleeding (n = 6), and cerebral infarction (n = 1). Tracheas were rinsed in ice-cold and sterile phosphate-buffered saline (PBS) to remove mucus and debris, opened longitudinally along the anterior surface, and mounted in a stretched position in a dissection tray. The surface epithelium was scored into longitudinal strips and was pulled off of the submucosa (34). The tissue strips were rinsed four times in PBS containing 5 mM dithiothreitol and then two times in PBS alone. The tissue strips were placed into the conical tubes (Costar, Cambridge, MA) containing protease (0.4 mg/ml; Sigma type XIV) dissolved in PBS. The strips were stored overnight in the refrigerator at 4°C. The enzyme was then competitively inhibited by the addition of FCS to a final concentration of 2.5%, and small sheets of cells were dislodged from the epithelial strips by vigorous agitation. The denuded strips were removed, and the sheets of cells remaining were dispersed by repeated aspiration in a 10-ml pipette.

Cells were pelleted (200 g, 10 min) and suspended in DMEM-Ham's F-12 containing 5% FCS (50:50 vol/vol). Cell counts were made using a hemocytometer, and estimates of viability were done using trypan blue and by measuring the amount of lactate dehydrogenase (LDH) in the medium as previously reported (2). The cells were then plated at 5 × 105 viable cells/ml in glass tubes coated with human placental collagen (34). This medium was replaced by DMEM-Ham's F-12 containing 2% USG on the first day after plating. The glass tubes were sealed with rubber plugs and were cultured at 37°C. Cell culture media were supplemented with 105 U/l penicillin, 100 mg/l streptomycin, 50 mg/l gentamicin, and 2.5 mg/l amphotericin B.

We screened 16 kinds of viruses (e.g., influenza types A, B, and C, parainfluenza virus, adenovirus, rhinovirus, and respiratory syncytial virus) in supernatants of cultured human tracheal epithelial cells before rhinovirus infection using the method described previously (20) and used the epithelial cell sheets without contamination by any viruses. Furthermore, we confirmed cilia beating on the epithelial cells and the absence of fibroblasts in glass tubes using the inverted microscope (MIT-2; Olympus, Tokyo, Japan). Finally, 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 using Ussing chamber methods (34). When cells cultured under these conditions become differentiated and form tight junctions without contamination of fibroblasts, they have values of >40 Omega  · cm2 for resistance and >10 µA/cm2 for short-circuit current (34). 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 Millicell CM inserts had high resistance (>40 Omega  · cm2) and high short-circuit current (>10 µA/cm2).

Viral stocks. HRV-2 and HRV-14 were prepared in our laboratory from the patients with common colds (20). Stocks of HRV-14 and HRV-2 were generated by infecting human embryonic fibroblast cells cultured in glass tubes in 1 ml of MEM supplemented with 2% GGFCS, 50 U/ml penicillin, and 50 µg/ml streptomycin at 33°C. The cells were incubated for several days in glass tubes in 1 ml of MEM supplemented with 2% GGFCS until 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 viral stock solutions was determined using the human embryonic fibroblast cell assay described below.

Detection and titration of viruses. Rhinoviruses were detected by exposing confluent human embryonic fibroblast cells in glass tubes to serial 10-fold dilutions of virus-containing medium or lysates in MEM supplemented with 2% GGFCS. Glass tubes were then incubated at 33°C for 7 days, and the cytopathic effects of viruses on human embryonic fibroblast cells were observed using an inverted microscope (MIT; Olympus) as reported previously (20). The amount of specimen required to infect 50% of human embryonic fibroblast cells (TCID50) was determined.

Viral infection of human tracheal epithelial cells. Medium was removed from confluent monolayers of human tracheal epithelial cells and was replaced with 1 ml of DMEM-Ham's F-12 containing 2% USG. Rhinovirus was added at a concentration of 105 TCID50/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 fed with DMEM-Ham's F-12 containing 2% USG supplemented with 105 U/l penicillin, 100 mg/l streptomycin, 50 mg/l gentamicin, and 2.5 mg/l amphotericin B and were cultured at 33°C with rolling in the incubator (HDR-6-T; Hirasawa, Tokyo, Japan). Supernatants were removed at various times after infection and were stored at -80°C for the determination of viral content. Viral titers of the material used for infection and of the supernatants removed at the end of the infection period were also determined to estimate the maximal amount of viral uptake that had occurred during the exposure period. As an additional control for nonspecific adherence of virus, the content of inocula after a 1-h incubation in empty glass tubes was also assessed. Finally, cell-associated viral content was analyzed using sonicated human tracheal epithelial cells. Viral content in the supernatant and cell-associated viral content are expressed as TCID50 units per milliliter and as TCID50 units per 106 cells, respectively.

Effects of antibodies to ICAM-1 on rhinovirus infection. Confluent human tracheal epithelial cells were incubated for 30 min at 37°C with medium alone, with medium containing either of the two mouse monoclonal anti-human antibodies to ICAM-1 [84H10 (100 µg/ml; Immunotech, Marseille, France) or RR1 (100 µg/ml; a gift from Boehringer Ingelheim, Ridgefield, CT; see Ref. 14)], or with medium containing an isotype-matched mouse immunoglobulin G1 (IgG1) control monoclonal antibody (100 µg/ml; Chemicon International). Both 84H10 and RR1 are IgG1 isotypes and recognize the ICAM-1 functional domain. After excess antibodies were washed off, the monolayers were exposed to HRV-2 (105 TCID50/ml) or HRV-14 (105 TCID50/ml) for either 15 or 60 min before rinsing and adding fresh DMEM-Ham's F-12 containing 2% USG supplemented with 105 U/l penicillin, 100 mg/l streptomycin, 50 mg/l gentamicin, and 2.5 mg/l amphotericin B. The viral content of this medium was then assessed at various times after infection.

Detection of rhinovirus RNA and cytokine mRNA by reverse transcription-polymerase chain reaction. Human tracheal epithelial cells cultured in the glass tubes were lysed by the addition of RNazol (0.2 ml/106 cells; BIOTECX, Houston, TX) and were transferred into Eppendorf tubes. The cell homogenates were mixed with a 10% volume of chloroform, shaken vigorously for 15 s, placed on ice for 15 min, and centrifuged at 12,000 g for 15 min at 4°C. The upper aqueous phase containing RNA was collected and mixed with an equal volume of isopropanol. Pellets of RNA were obtained by centrifugation at 12,000 g for 15 min at 4°C, dissolved in a water, and stored at -80°C before use.

Polymerase chain reaction (PCR) was performed as previously described (4, 10). Briefly, 2 µg of RNA from each aliquot of human tracheal epithelial cells were dissolved in a 100-µl buffer containing the reagents for the reverse transcriptase (RT) reaction with the following composition: 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 5 U/µl Moloney murine leukemia virus RT (GIBCO-BRL Life Technologies), 0.5 mM deoxynucleoside 5'-triphosphate (dNTP; Takara, Ohtsu, Japan), 1 U/µl ribonuclease inhibitor (Promega, Madison, WI), and 5 µM random hexamers (Pharmacia Biotech, Uppsala, Sweden). The RT reaction was performed for 60 min at 37°C, followed by 95°C for 10 min. The resulting cDNA was frozen at -80°C until use in the PCR. For each sample, 5 µl of RT mixture were added to a 45-µl PCR mixture consisting of 10 mM Tris · HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, and 1.25 units Taq polymerase (Takara). Primer pairs for the rhinovirus and cytokines were present at 2 ng/µl. Sequences of the PCR primer pairs used in these experiments are shown in Table 1. The PCR was performed in an automated thermal cycler (MJ Research, Watertown, MA), and 10 µl of the reaction were removed at 30 cycles for each sample. Samples were separated on a 2% agarose gel (FMC BioProducts, Rockland, ME) and were stained for 30 min in 1 µg/ml ethidium bromide. The DNA bands were visualized on a ultraviolet illuminator and were photographed with type 667 positive/negative film (Polaroid, Cambridge, MA).

                              
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Table 1.   Polymerase chain reaction primer sequences

For rhinovirus and each cytokine, mRNA expressions in human tracheal epithelial cells were examined before and at 1, 3, and 5 days after HRV-14 infection.

Cytokine assays. In the preliminary study, we found that the mRNA of IL-1beta , IL-6, IL-8, TNF-alpha , and GM-CSF were expressed in cultured human tracheal epithelial cells before and after HRV-14 infection. To determine the effects of HRV-2 or HRV-14 infection on the production of cytokines, we measured the amount of IL-1beta , IL-6, IL-8, and TNF-alpha released from human tracheal epithelial cells into the culture medium before and at 1, 3, and 5 days after HRV-2 or HRV-14 infection. We also measured the amount of GM-CSF and IL-1alpha in the supernatants of human tracheal epithelial cells before and after HRV-14 infection and the amount of IL-1beta and TNF-alpha in the viral stocks. Proteins of IL-1alpha , IL-1beta , IL-6, IL-8, TNF-alpha , and GM-CSF were measured by specific enzyme-linked immunosorbent assays (ELISA). Sensitivities of the assays were 10 pg/ml for the IL-1alpha and IL-1beta ELISA kit (Ohtsuka, Tokushima, Japan), the IL-6 ELISA kit (Toray, Tokyo, Japan), and the IL-8 ELISA kit (Toray), 4 pg/ml for the TNF-alpha ELISA kit (Ohtsuka), and 2 pg/ml for the GM-CSF ELISA kit (Genzyme, Cambridge, MA). We used an average value of replicate cultures from the same trachea (n = 3) for the analysis of cytokine production.

To determine the effects of antibodies to ICAM-1 on production of cytokines induced by HRV-2 or HRV-14, confluent human tracheal epithelial cells were incubated for 30 min with medium alone or with a medium containing either 84H10 (100 µg/ml) or RR1 (100 µg/ml) at 37°C before HRV-2 or HRV-14 infection. We also tested the effects of an isotype-matched mouse IgG1 control monoclonal antibody (Chemicon International) at the same concentration as antibodies to ICAM-1 on production of cytokines induced by HRV-2 or HRV-14.

Northern blot analysis. Northern blot analysis was done as described previously (26). Equal amounts of total RNA (10 µg) extracted from human tracheal epithelial cells, as determined spectrophotometrically, were subjected to electrophoresis in 1% agarose-formaldehyde gel. The gel was then transferred via capillary action onto a nylon membrane (Hybond-N+; Amersham Life Science). The membrane was hybridized with [alpha -32P]dCTP (3,000 Ci/mmol; Amersham)-labeled human ICAM-1 cDNA (1.8-kb Xba I fragment; British Bio-technology, Oxon, UK) with a random-prime labeling kit (Random Primer; Takara). Hybridization with a radiolabeled probe was performed overnight at 42°C. After high-stringency washing was performed (1× standard saline citrate, 0.1% sodium dodecyl sulfate, 60°C), autoradiographic detection of the hybridized probe was performed by exposing Kodak Scientific Imaging film for 48-72 h at -70°C. Quantitation of autoradiographic bands was accomplished with an image analyzer (Bio Imaging Analyzer, BAS-2000; Fuji Photo Film) and was expressed as the intensity of the ICAM-1/beta -actin bands. We used an average value of replicate cultures from the same trachea (n = 3) for analysis of the intensity of the ICAM-1/beta -actin bands.

To study the effects of HRV-2 or HRV-14 infection on the mRNA expression of ICAM-1 in human tracheal epithelial cells, cells were examined 1, 3, and 5 days after HRV-14 infection and 5 days after HRV-2 infection.

To determine the mechanisms responsible for upregulation of ICAM-1 mRNA expression after HRV-14 infection, we tested the effects of either IL-1beta (200 pg/ml; Ohtsuka), IL-6 (100 pg/ml; Genzyme), IL-8 (100 pg/ml; Collaborative Research, Bedford, MA), or TNF-alpha (10 pg/ml; Genzyme) on ICAM-1 mRNA expression in human tracheal epithelial cells. The concentration of each cytokine chosen was matched to a net increase in the culture medium after HRV-14 infection, and cells were incubated overnight with each cytokine.

Effects of neutralizing antibodies to IL-1beta and TNF-alpha on HRV-14 infection and ICAM-1 mRNA expression. To determine the role of endogenous IL-1beta in viral infection and ICAM-1 expression, confluent human tracheal epithelial cells were preincubated using a monoclonal mouse anti-human IL-1beta (10 µg/ml; Genzyme) or an isotype-matched mouse IgG1 control monoclonal antibody (Chemicon International), at the same concentration for 5 days. We also tested the effects of a monoclonal mouse anti-human TNF-alpha (10 µg/ml, 5 days; Genzyme) on viral infection and ICAM-1 expression. Viral titers in the supernatants collected during 3-5 days and the expression of ICAM-1 mRNA 5 days after HRV-14 infection (105 TCID50/ml) were measured in confluent human tracheal epithelial cells preincubated with each antibody.

Effects of IL-1beta on susceptibility to HRV-14 infection. To examine whether IL-1beta increases the susceptibility to HRV-14 infection, confluent human tracheal epithelial cells were preincubated with or without IL-1beta (200 pg/ml) for 24 h. The epithelial cells were then exposed to serial 10-fold dilutions of HRV-14 for 1 h at 33°C. The presence of HRV-14 in supernatants collected during 1-3 days after infection was determined using the human embryonic fibroblast cell assay described above to assess whether infection occurred at each dose of HRV-14 used. This index of susceptibility to infection, defined as the minimum dose of HRV-14 that could induce infection, was compared with the susceptibility of control cells that were not preincubated with IL-1beta (27).

Immunohistochemical analysis. Immunohistochemical analysis for ICAM-1 expression in human tracheal epithelial cells was done as described previously (21). Human tracheal epithelial cells were cultured on Vitrogen gels on Millicell inserts (0.45 µm pore size and 0.6 cm2 area; Millipore Products Division; see Ref. 34). Cell sheets were fixed in periodate-lysine-paraformaldehyde at 4°C for 2 h. After a wash in sucrose (10, 15, or 20%)-PBS, they were embedded in optimum cutting temperature compound (Miles Laboratories, Naperville, IL) in liquid nitrogen and were stored at -70°C until use. The staining was performed using an alkaline phosphatase-anti-alkaline phosphatase (APAAP) method. Cryostat sections (6 µm) were incubated with 75% heat-inactivated normal human AB serum (Sigma) in Tris-buffered saline (pH 7.6) containing 0.05 M Tris base, 0.15 M NaCl, 0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 0.01% saponin (TBS[H + S]; Sigma) for 1 h at room temperature and then with 20% normal rabbit serum (Dako) in TBS[H + S] for 20 min at room temperature to block nonspecific binding of the first and second antibodies. The sections were overlaid with 4 µg/ml of the antibody 84H10 (Immunotech) or with an isotype-matched mouse monoclonal antibody with the irrelevant specificity (Chemicon International) in TBS[H + S] containing 1% bovine serum albumin (TBS[H + S]-BSA, fraction V; Sigma). After overnight incubation at 4°C, the sections were incubated with anti-mouse immunoglobulin rabbit immunoglobulins (Dako) diluted 40 times with TBS[H + S]-BSA for 30 min at room temperature followed by incubation with soluble complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase (Dako) diluted 60 times with TBS[H + S]-BSA for 30 min at room temperature. After one more repeat of these procedures, the sections were developed by exposure to substrate for 8 min with the fast red substrate system (Dako) according to the manufacturer's protocol and were counterstained with hematoxylin.

To study the effects of HRV-14 infection on ICAM-1 expression in human tracheal epithelial cells, HRV-14 was added at a concentration of 105 TCID50/ml to the mucosal side of cell sheets cultured on Millicell inserts. After a 1-h incubation at 33°C, the viral solution was removed, and both sides of the cell sheets were rinsed with PBS. Before immunohistochemical studies, cell sheets were cultured for 2 days at 33°C.

We also tested the effects of either IL-1beta (200 pg/ml; Ohtsuka), IL-6 (100 pg/ml; Genzyme), IL-8 (100 pg/ml; Collaborative Research), or TNF-alpha (10 pg/ml; Genzyme) on ICAM-1 expression in human tracheal epithelial cells. Cells were incubated overnight with each cytokine. In addition, we tested a 10 ng/ml concentration of IL-1beta as a positive control (29).

Statistical analysis. Results are expressed as means ± SE. Statistical analysis was performed using a one-way analysis of variance (ANOVA), and multiple comparisons were made using Bonferroni's method. Significance was accepted at P < 0.05; n refers to the number of donors from which cultured epithelial cells were used.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Rhinovirus infection of human tracheal epithelial cells. Exposing confluent human tracheal epithelial cell monolayers to HRV-2 or HRV-14 (105 TCID50/ml) consistently led to infection. Collection of culture medium at differing times after viral exposure revealed no detectable virus at 1 h after infection. Both HRV-2 and HRV-14 were detected in 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 the demonstration that the viral titers of supernatants collected during 1-3 days, 3-5 days, and 5-7 days after infection each contained significant levels of HRV-2 or HRV-14 (Fig. 1B). Analysis of the levels of cell-associated virus (the virus detectable in sonicates of the human tracheal epithelial cells) followed a similar time course to that observed in the medium. The viral titers of cell-associated HRV-14 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 ± 0.3 log TCID50 units at 24 h, 2.5 ± 0.3 log TCID50 units at 3 days, 2.2 ± 0.3 log TCID50 units at 5 days, and 1.6 ± 0.3 log TCID50 units at 7 days (n = 7 each). In both cell supernatants and lysates, viral titer levels increased significantly with time (P < 0.05 in each case by ANOVA). Viral titers at 24 h after HRV-14 infection in cells from smokers did not differ significantly from those in nonsmokers (3.2 ± 0.3 log TCID50 units in smokers vs. 3.3 ± 0.3 log TCID50 units in nonsmokers; P > 0.50). Human tracheal cell viability, as assessed by the exclusion of trypan blue, was consistently >96% in HRV-14-infected culture. Likewise, HRV-14 infection did not alter the amount of LDH in the supernatants (29 ± 3 IU/l before vs. 33 ± 3 IU/l 5 days after infection; P > 0.20, n = 7). HRV-14 infection also had no effect on cell numbers. Cell counts 24 h after infection were not significantly different (1.7 ± 0.1 × 106 in noninfected cells vs. 1.7 ± 0.2 × 106 in infected cells; P > 0.50, n = 7).


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Fig. 1.   Viral titers in supernatants of human tracheal epithelial cells obtained at different times after exposure to 105 TCID50/ml of human rhinovirus (HRV)-14 (open circle ) and HRV-2 (bullet ). TCID50 is the amount of specimen required to infect 50% of human embryonic fibroblast cells. A: viral titers in supernatants collected at sequential times during the first 24 h after infection. B: viral titers of HRV-14 (open bars) and HRV-2 (filled bars) in supernatants collected during 1-3 days, 3-5 days, and 5-7 days after infection. Results are reported as means ± SE from 7 samples.

Detection of viral RNA by PCR. Further evidence of HRV-14 infection of human tracheal epithelial cells and of viral replication was provided by PCR analysis (Fig. 2). In each of three experiments, RNA extracted from uninfected cells did not produce any detectable PCR product at 381 bp (0 h). A faint product was observable in RNA extracted from cells 6 h after infection followed by a progressive increase in viral RNA until 3 days after infection.


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Fig. 2.   Time course of replication of rhinovirus RNA from human tracheal epithelial cells after HRV-14 infection detected by reverse transcription-polymerase chain reaction. beta -Actin was used as a housekeeping gene. M, phi X174/Hinc II fragment molecular weight markers. Data are representative of 3 different experiments.

Effects of rhinovirus infection on cytokine production. Human tracheal epithelial cells were screened for mRNA expression of various cytokines. PCR analysis revealed mRNA expression for IL-1beta , IL-6, IL-8, TNF-alpha , and GM-CSF before and after cells were exposed to HRV-14 (105 TCID50/ml). However, mRNA for IL-4, IL-5, IL-10, and interferon-gamma were not detectable in human tracheal epithelial cells before and after HRV-14 infection in all seven experiments (data not shown). Figure 3 shows the time course of IL-1beta , IL-6, IL-8, and TNF-alpha production in supernatants of human tracheal epithelial cells after HRV-14 (Fig. 3A) or HRV-2 (Fig. 3B) infection. Because viral infection did not alter cell numbers (see above), all cytokine values are reported in picograms per milliliter of supernatant. Basal secretion was quite high with IL-8, relatively high with IL-6, but low or negligible with IL-1beta and TNF-alpha . However, secretion of IL-1beta , IL-6, IL-8, and TNF-alpha all increased in response to both HRV-2 and HRV-14, although, in terms of absolute levels, IL-1beta (195 ± 15 pg/ml in HRV-2 and 145 ± 15 pg/ml in HRV-14) and IL-6 (185 ± 22 pg/ml in HRV-2 and 120 ± 14 pg/ml in HRV-14) predominated. Of the cytokines measured, GM-CSF and IL-1alpha levels were not changed by HRV-14 infection (140 ± 10 pg/ml before vs. 165 ± 18 pg/ml after in GM-CSF and 12 ± 2 pg/ml before vs. 13 ± 2 pg/ml after in IL-1alpha ; P > 0.20, n = 7).


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Fig. 3.   Time course of release of cytokines into supernatants of human tracheal epithelial cells after HRV-14 (A) and HRV-2 (B) infection (bullet ). (open circle ) Sham infection (control). IL, interleukin; TNF-alpha , tumor necrosis factor-alpha . Results are reported as means ± SE from 7 samples. Significant differences from corresponding control values are indicated by * P < 0.05 and ** P < 0.01.

In contrast to supernatants of human tracheal epithelial cells, neither IL-1beta nor TNF-alpha was detectable in viral stocks.

Effects of antibodies to ICAM-1 on rhinovirus infection and cytokine production. Incubation of cells with both mouse monoclonal antibodies to ICAM-1 completely blocked HRV-14 infection, as assessed by the absence of detectable viral titers in the supernatants recovered 24 h after 15 min of HRV-14 exposure (2.2 ± 0.2 log TCID50 units in control, 0 ± 0 log TCID50 units in 84H10, and 0 ± 0 log TCID50 units in RR1). Likewise, viral titers 24 h after 60 min of HRV-14 exposure were significantly decreased by 84H10 (1.7 ± 0.3 log TCID50 units; P < 0.01, n = 7) and RR1 (1.9 ± 0.3 log TCID50 units; P < 0.01, n = 7) from control values (3.3 ± 0.1 log TCID50 units; n = 7). These treatments also significantly inhibited increases in IL-1beta , IL-6, IL-8, and TNF-alpha production induced by HRV-14 infection (Fig. 4A). However, an isotype-matched IgG1 control monoclonal antibody failed to alter viral titers in the supernatants 24 h after 15 min of viral exposure (2.3 ± 0.2 log TCID50 units; P > 0.50, n = 7) and 60 min of viral exposure (3.3 ± 0.2 log TCID50 units; P > 0.50, n = 7). Likewise, IgG1 control monoclonal antibody did not inhibit increases in IL-1beta , IL-6, IL-8, and TNF-alpha production induced by HRV-14 infection (Fig. 4A). In contrast to HRV-14, viral titers in the supernatants recovered 24 h after 15 min of HRV-2 exposure were not altered by 84H10 (2.4 ± 0.2 log TCID50 units; P > 0.50, n = 7) and RR1 (2.3 ± 0.2 log TCID50 units; P > 0.50, n = 7) from control values (2.3 ± 0.2 log TCID50 units; n = 7) and from values of IgG1 control monoclonal antibody treatment (2.2 ± 0.3 log TCID50 units; n = 7). Neither 84H10, RR1, nor IgG1 control monoclonal antibody altered increases in production of IL-1beta , IL-6, IL-8, and TNF-alpha induced by HRV-2 infection (Fig. 4B).


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Fig. 4.   Release of cytokines into supernatants of human tracheal epithelial cells in the presence of monoclonal antibodies of RR1 (filled bars) and 84H10 (stippled bars) to intercellular adhesion molecule-1 (ICAM-1) mouse purified immunoglobulin G1 (IgG1) monoclonal antibody (hatched bars) or absence of an antibody (control; open bars). Results are reported as means + SE from 7 samples. Significant differences from viral infection alone are * P < 0.05 and ** P < 0.01. Effects of monoclonal antibodies to ICAM-1 and mouse IgG1 monoclonal antibody were examined at the maximal production of each cytokine after HRV-14 (A) and HRV-2 (B) infection.

Effect of rhinovirus infection on ICAM-1 mRNA expression. The baseline expression of ICAM-1 was constant in confluent human tracheal epithelial cell sheets, and the coefficient of variation was small (9.8%; n = 22). Neither smoking habit nor cause of death influenced the baseline expression of ICAM-1 mRNA. Exposure of human tracheal epithelial cells to HRV-14 (Fig. 5A) or HRV-2 (Fig. 5B) caused increases in ICAM-1 mRNA compared with sham exposure (control). Human tracheal epithelial cells 5 days after HRV-14 (Fig. 5C) or HRV-2 (Fig. 5D) infection were shown to overexpress ICAM-1 mRNA twofold compared with those 5 days after a sham exposure. IL-1beta (200 pg/ml) increased ICAM-1 mRNA to the levels similar to those induced by rhinovirus infection expressed as the intensity of the ICAM-1/beta -actin bands (0.49 ± 0.03 scan units; P < 0.01, n = 7). However, neither IL-6 (100 pg/ml), IL-8 (100 pg/ml), nor TNF-alpha (10 pg/ml) altered the levels (0.23 ± 0.03 scan units in IL-6, 0.22 ± 0.03 scan units in IL-8, and 0.21 ± 0.03 scan units in TNF-alpha ; P > 0.20, n = 7) compared with sham exposure (0.21 ± 0.02 scan units; n = 7).


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Fig. 5.   Northern blot analysis demonstrating increases in ICAM-1 mRNA levels of human tracheal epithelial cells 1, 3, and 5 days after HRV-14 infection (A) and 5 days after HRV-2 infection (B) compared with sham infection (control). beta -Actin was used as a housekeeping gene. C and D: expression of ICAM-1 mRNA in human tracheal epithelial cells 5 days after HRV-14 infection (C) and HRV-2 infection (D; filled bars). Open bars, sham infection (control). ICAM-1 mRNA is normalized to constitutive expression of beta -actin mRNA. Results are reported as means + SE from 7 samples. Significant differences from corresponding control values are indicated by ** P < 0.01.

Effects of neutralizing antibodies to IL-1beta and TNF-alpha on viral infection and ICAM-1 mRNA expression. The monoclonal mouse anti-human IL-1beta (10 µg/ml) significantly decreased HRV-14 titers of supernatant collected during 3-5 days (Fig. 6A) and inhibited ICAM-1 mRNA expression in human tracheal epithelial cells (Fig. 6B). In contrast, neither the monoclonal mouse anti-human TNF-alpha (10 µg/ml) nor mouse IgG1 control monoclonal antibody (10 µg/ml) altered viral titers of supernatant (Fig. 6A) and ICAM-1 mRNA expression (Fig. 6B).


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Fig. 6.   HRV-14 titers in supernatants collected during 3-5 days (A) and expression of ICAM-1 mRNA (B) in human tracheal epithelial cells 5 days after HRV-14 infection in the presence of neutralizing antibodies to IL-1beta (a-IL-1beta ; 10 µg/ml; filled bars), TNF-alpha (a-TNF-alpha ; 10 µg/ml; stippled bars), or mouse IgG1 monoclonal antibody (IgG1; 10 µg/ml; hatched bars) and absence of an antibody (control; open bars). ICAM-1 mRNA is normalized to constitutive expression of beta -actin mRNA. Results are reported as means + SE from 7 samples. Significant differences from HRV-14 infection alone are indicated by ** P < 0.01.

Effects of IL-1beta on susceptibility to HRV-14 infection. Pretreatment of the human tracheal epithelial cells for 24 h with IL-1beta (200 pg/ml) increased the susceptibility of cells to HRV-14 infection, decreasing by 10-fold the minimum dose of virus necessary to cause infection (1.1 ± 0.1 log TCID50 units in IL-1beta vs. 2.2 ± 0.2 log TCID50 units in control; P < 0.01, n = 7).

Immunohistochemical analysis. Figure 7 shows ICAM-1 expression in the human tracheal epithelial cells, which was detected as a red color. HRV-14 infection (Fig. 7B) increased ICAM-1 expression in both apical and basolateral sides compared with sham infection, which caused staining only on the basolateral side (Fig. 7A). Treatment with IL-1beta at either 200 pg/nl (Fig. 7C) or 10 ng/ml (Fig. 7D) mimicked HRV-14 infection, with a stronger staining observed in the latter. In contrast, treatment with either IL-6, IL-8, or TNF-alpha failed to alter ICAM-1 expression in the human tracheal epithelial cells (not shown).


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Fig. 7.   ICAM-1 expression in human tracheal epithelial cells cultured for 2 days after sham infection (A; control) or HRV-14 infection (B) and that treated with either 200 pg/ml (C) or 10 ng/ml (D) concentration of IL-1beta . Cells expressing ICAM-1 are stained as red and are indicated by arrows (bar = 20 µm and magnification = ×100).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Primary cultures of human airway epithelial cells are thought to be important for characterizing viral infection in the airway and for advancing our knowledge of airway inflammation (8). Respiratory viral infection of primary cultures of human nasal epithelial cells were reported previously, but these cultures contained a significant number of fibroblasts (32), making it difficult to interpret the results obtained. We report rhinovirus infection of primary cultures of the human tracheal epithelial cells that do not contain other cell types such as fibroblasts (34). However, it should be noted here that this model system has significant differences from airway epithelial cells in vivo.

Viral infection of cultured human tracheal epithelial cells and subsequent viral replication were confirmed by showing the increased viral content in the culture medium of infected cells with time, assessed by the cytopathic effects of this medium on human embryonic fibroblast cells, and by showing that the cytopathic effects of human tracheal epithelial cell lysates also increased with time after infection. Viral replication was also detected by PCR of viral RNA after reverse transcription into DNA. A progressive increase in viral RNA, observed until 3 days after infection, was detected by a pronounced band on PCR compared with the absence of any signal in RNA extracted from uninfected cells. Infections of human tracheal epithelial cells with HRV-2 and HRV-14 were consistently observed when confluent monolayers were exposed to virus. However, HRV-14 infection failed to influence both cell numbers and cell viability. This is in agreement with previous studies showing the lack of cytotoxicity on epithelial cells in rhinovirus infection (27, 32).

Infection of the respiratory epithelium by viruses has been shown to cause increased production of cytokines (19, 27). Subauste et al. (27) demonstrated that infection of BEAS-2B cells with HRV-14 induced an increased production of IL-6, IL-8, and GM-CSF. The present study is in agreement with a previous study showing the enhanced production of IL-6 and IL-8 within 24 h after infection (27). However, infection of human tracheal epithelial cells with HRV-2 and HRV-14 also caused increases in the production of IL-1beta and TNF-alpha , which differed significantly from HRV-14 infection of BEAS-2B cells (27). Although an increase in TNF-alpha production after infection was subtle and that in IL-8 was <30% from the baseline, there was a large amount of IL-1beta production from human tracheal epithelial cells in the present study, which was maximal at 3 days and was sustained up to 5 days after infection. IL-1beta is a potent inflammatory cytokine that induces growth and differentiation of T and B lymphocytes, other cytokine productions, prostaglandin E2 synthesis, and degranulation from neutrophils (1). IL-1beta also causes increases in ICAM-1 expression on both epithelial and vascular endothelial cells (1, 5, 29). Increases in ICAM-1 expression on vascular endothelial cells promote the adhesion of neutrophils, monocytes, and lymphocytes to these cells (5). Likewise, upregulation of ICAM-1 on HeLa cells is associated with the increased binding of major group rhinoviruses (28).

The specificity of the infection process for primary cultures of human tracheal epithelial cells by HRV-14 was confirmed by demonstrating that infection could be blocked using antibodies to ICAM-1 but not by an isotype-matched IgG1 monoclonal antibody. Furthermore, antibodies to ICAM-1 failed to block HRV-2 infection, a minor group of rhinoviruses that do not use ICAM-1 as its receptor. However, inhibition became less consistent at longer incubation times (e.g., 1 h), presumably because of the high affinity of the virus for its receptor and of the requirement for very few viral particles to enter the cell to induce infection (27). In addition, we showed that antibodies to ICAM-1 significantly inhibited the production of IL-1beta , IL-6, IL-8, and TNF-alpha induced by HRV-14 but that HRV-2-induced effects on cytokine production were not altered by antibodies to ICAM-1. However, antibodies to ICAM-1 could not achieve complete inhibition of cytokine production induced by HRV-14 infection, which may be also due to longer incubation times described above. Furthermore, we showed that rhinovirus infection and IL-1beta at the experimentally measured concentration in supernatants upregulated ICAM-1 expression assessed by increases in ICAM-1 mRNA using Northern blot analysis, whereas an increase in ICAM-1 mRNA was two times the control and relatively small compared with the study on respiratory syncytial virus infection of human pulmonary type II-like epithelial (A549) cells (22). Similar to ICAM-1 mRNA levels, however, both HRV-14 infection and IL-1beta increased ICAM-1 expression on epithelial cells as shown by immunohistochemical analysis, and IL-1beta increased susceptibility to HRV-14 infection. Furthermore, enhancement of ICAM-1 mRNA expression by HRV-14 infection was almost entirely blocked by the antibody to IL-1beta , and the anti-IL-1beta antibody significantly inhibited HRV-14 infection, confirming the role of the endogenous IL-1beta in viral infection and ICAM-1 expression in the present study. Although TNF-alpha is reported to upregulate the expression of ICAM-1 on epithelial cells (27), the present study failed to show the expression. Furthermore, the antibody to TNF-alpha did not alter HRV-14 infection or ICAM-1 mRNA expression. Lack of TNF-alpha -induced effects on ICAM-1 mRNA expression is probably due to a small amount of TNF-alpha production from human tracheal epithelial cells in response to HRV-14 infection. Although IL-1alpha is reported to be induced by A549 cells in response to respiratory syncytial virus infection (22), HRV-14 did not alter IL-1alpha production in the present study. The discrepancy may be explained by differences in the species of virus and cultured cells.

Upregulation of ICAM-1 expression on epithelial cells and the production of cytokines from these cells in response to rhinovirus infection may be relevant to the pathogenesis of airway inflammation associated with colds and mechanisms of viral exacerbations of asthma. IL-6 induces antibody production in B cells and T cell activation and differentiation (1). IL-8 is a major chemoattractant for neutrophils and stimulates neutrophils to cause enzyme release and production of reactive oxygen metabolites (12). Neutrophils and lymphocytes are shown to be predominant cell types in the nasal mucosa during rhinovirus infection (13). Likewise, upregulation of ICAM-1 could increase susceptibility to major group rhinoviruses (7, 28) and could lead cells adjacent to infected cells to infection when viruses are released from the cells originally infected. Furthermore, chronic antigen challenge is shown to increase ICAM-1 expression on airway epithelium, which may be related to airway inflammation in asthma (31). Inflammatory conditions such as asthma, smoking, and ozone exposure in which ICAM-1 expression is increased on respiratory epithelial surfaces may cause a predisposition to rhinovirus infection through increasing expression of the major group of rhinovirus receptors. The rhinovirus infection would enhance airway inflammation by recruiting neutrophils and, potentially, other inflammatory cells, causing increased mediator release and exacerbation of the underlying reactive airway diseases.

In summary, we have shown, for the first time, that rhinovirus is infectious in primary cultures of human tracheal epithelial cells. Rhinovirus infection upregulates ICAM-1 expression on epithelial cells. Rhinovirus infection induces an increased production of cytokines that regulate the acute phase reaction of airway inflammation. Of these, IL-1beta is able to upregulate ICAM-1 expression and to increase the susceptibility to HRV-14 infection, and the antibody to IL-1beta inhibited both viral infection and ICAM-1 expression. Furthermore, antibodies to ICAM-1 reduce the production of cytokines induced by HRV-14 infection. These findings suggest that rhinoviruses per se amplify their infection by the overexpression of ICAM-1 on epithelial cells through the production of IL-1beta , resulting in a further increase in the production of inflammatory cytokines. A recent report has also demonstrated a similar IL-1alpha -dependent autocrine mechanism in respiratory syncytial virus infection of A549 cells (22). Thus these processes may be relevant to airway inflammation induced by respiratory viruses and viral exacerbations of asthma.

    ACKNOWLEDGEMENTS

We thank Grant Crittenden for English correction and Akira Ohmi, Michiko Okamoto, and Minako Tada for technical assistance.

    FOOTNOTES

Address for reprint requests: H. Sasaki, Dept. of Geriatric Medicine, Tohoku University School of Medicine, Aoba-ku Seiryo-machi 1-1, Sendai 980, Japan.

Received 16 September 1996; accepted in final form 24 June 1997.

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Discussion
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