Type 2 rhinovirus infection of cultured human tracheal epithelial cells: role of LDL receptor

Tomoko Suzuki1, Mutsuo Yamaya1, Masahito Kamanaka1, Yu X. Jia1, Katsutoshi Nakayama1, Masayoshi Hosoda1, Norihiro Yamada1, Hidekazu Nishimura3, Kiyohisa Sekizawa2, 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
METHODS
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DISCUSSION
REFERENCES

To examine the role of the low-density lipoprotein (LDL) receptor on minor group human rhinovirus (RV) infection, primary cultures of human tracheal epithelial cells were infected with a minor group (RV2) or a major group (RV14) RV. Viral infection was confirmed by showing with PCR that viral titers in supernatants and lysates from infected cells increased with time. RV2 and RV14 increased expression of mRNA and protein of the LDL receptor on the cells and the cytokine production. RV2 induced activation of transcription factors SP1 and nuclear factor-kappa B (NF-kappa B). An antibody to the LDL receptor inhibited RV2 infection and RV2-induced cytokine production without an effect on RV14 infection and RV14-induced cytokine production. These findings imply that RV2 upregulates LDL receptor expression on airway epithelial cells, thereby increasing susceptibility to minor group RV infection. LDL receptor expression and cytokine production may be mediated, in part, via activation of transcription factors by RV2. These events may be important in airway inflammation after minor group RV infection in asthma.

asthma; common cold; airway inflammation; low-density lipoprotein receptor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RHINOVIRUSES (RVs) cause the majority of common colds, which often provoke wheezing in patients with asthma (18), and have also been associated with exacerbations of chronic bronchitis (14). A perspective study (17) has indicated that asthma attacks are associated with a viral infection in as many as 20-50% of the cases. Studies (12, 18) 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. 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 (29), but the clinical and pathological features of RV infection are, to a great extent, due to the elaboration by the host of a variety of inflammatory mediators (39).

The 102 antigenically distinct serotypes of RVs are divided into two groups, major and minor, based on receptor specificity (1, 35). The major group RVs bind to intercellular adhesion molecule-1 (ICAM-1) (9, 33). Airway epithelial cells express ICAM-1, and monoclonal antibodies to anti-ICAM-1 inhibit both infection by major group RVs and cytokine production induced by major group RV infection (31, 32). Because major group RVs upregulate ICAM-1 production in airway epithelial cells (23, 32), they are suggested to amplify their infection by the overexpression of ICAM-1 on epithelial cells, resulting in a further increase in the production of inflammatory cytokines (23, 31, 32). In contrast, a recent report (11) demonstrated that the low-density lipoprotein (LDL) receptor family can function as receptors for minor group RVs in human fibroblasts. RV type 2 (RV2), a minor group RV, can also infect airway epithelial cells and produce inflammatory cytokines (31, 32). However, the role of the LDL receptor in RV2 infection of the airway epithelial cells remains uncertain.

We therefore investigated whether primary cultures of human tracheal epithelial cells can be infected with minor group RV2 through binding to a LDL receptor. We also studied whether minor group RV2 infection upregulates LDL receptor expression on the airway epithelial cells and increases the susceptibility to infection.


    METHODS
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Medium 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 ultra-low IgG FCS were from GIBCO BRL (Life Technologies, Palo Alto, CA); trypsin, EDTA, dithiothreitol (DTT), Sigma type XIV protease, human placental collagen, penicillin, streptomycin, gentamicin, and amphotericin B were from Sigma (St. Louis, MO); and Ultroser G (USG) serum substitute was from BioSepra (Marlborough, MA).

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 (19) in a Roux-type bottle (Iwaki Glass, Chiba, Japan) sealed with a rubber plug. 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) were suspended in MEM containing 10% FCS and then plated in glass tubes (15 × 105 mm; Iwaki Glass) that were sealed with rubber plugs and kept stationary at a slant of 5° at 37°C.

Human tracheal epithelial cell culture. Tracheae for cell culture were obtained 2-8 h after death from 51 patients (age, 64 ± 5 yr; 22 women and 29 men) under a protocol passed by the Tohoku University (Sendai, Japan) Ethics Committee. Fifteen of the patients were smokers. None had a respiratory illness including bronchial asthma, and they died of acute myocardial infarction (n = 12), congestive heart failure (n = 5), malignant tumor other than lung cancer (n = 14), rupture of aortic aneurysm (n = 2), liver cirrhosis (n = 3), renal failure (n = 4), leukemia (n = 3), malignant lymphoma (n = 1), cerebral bleeding (n = 6), and cerebral infarction (n = 1).

Isolation and culture of the human tracheal surface epithelial cells were performed as previously described (20, 32, 38). 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) overnight at 4°C. The cells were pelleted (200 g for 10 min) and suspended in DMEM-Ham's F-12 medium containing 5% FCS (50:50 vol/vol). Cell counts were made with a hemacytometer, and estimates of viability were done with trypan blue and by measuring the amount of lactate dehydrogenase in the medium as previously reported (32). The cells were then plated at 5 × 105 viable cells/ml in round-bottom glass tubes (15-mm diameter and 105-mm length; Iwaki Glass) coated with human placental collagen (32, 38). This medium was replaced by DMEM-Ham's F-12 medium containing 2% USG on the first day after plating. The glass tubes were sealed with rubber plugs, kept stationary at a slant of 5°, and cultured at 37°C. The cell culture medium was supplemented with 105 U/l of penicillin, 100 mg/l of streptomycin, 50mg/l of gentamicin, and 2.5 mg/l of amphotericin B.

We confirmed cilia beating on the epithelial cells and the absence of fibroblasts in the glass tubes using an inverted microscope (MIT-2, Olympus, Tokyo, Japan) as previously described (32). Under an inverted microscope, the cultured human tracheal epithelial cells had a cuboidal or round-shaped appearance of confluent sheets. In contrast, the fibroblasts had a spindle-shaped appearance. We found in the preliminary experiments that RV infection caused cytopathic effects on the spindle-shaped fibroblasts as shown by Winther et al. (37) but not on the tracheal epithelial cells with a cuboidal or round-shaped appearance. 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 using Ussing chamber methods (20, 32, 38). 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 (20, 32, 38). Therefore, cultured human tracheal epithelial cells were judged as cells able to form tight junctions and were used for the following experiments when the cells on the Millicell CM inserts had a high resistance (>40 Omega  · cm2) and a high short-circuit current (>10 µA/cm2). In addition, we observed whether the human tracheal epithelial cells made a dome formation to confirm that cells on a solid glass support form tight junctions. We found that the human tracheal epithelial cells made a dome formation when the cells formed confluent cell sheets as described by Widdicombe et al. (36).

Viral stocks. RV2 and RV14 were prepared in our laboratory from patients with common colds (19). Stocks of RV2 and RV14 were generated by infecting human embryonic fibroblast cells cultured in 1 ml of MEM supplemented with 2% ultra-low IgG FCS, 5 × 104 U/l of penicillin, and 50 mg/l of streptomycin in glass tubes at 33°C. The cells were incubated for several days in glass tubes in 1 ml of MEM supplemented with 2% ultra-low IgG FCS instead of gamma -globulin-free calf serum (19, 32) until cytopathic effects were obvious, after which the cultures were frozen at -80°C, thawed, and sonicated. The virus-containing fluid 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. We found in the preliminary experiments that human embryonic fibroblast cells produce the same content of RV2 or RV14 in MEM supplemented with ultra-low IgG FCS as in MEM with gamma -globulin-free calf serum (GIBCO BRL) (19, 32).

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% ultra-low IgG FCS. The glass tubes were then incubated at 33°C for 7 days, and the cytopathic effects of the viruses on human embryonic fibroblast cells were observed with an inverted microscope (MIT, Olympus) as previously reported (19, 20, 32). The amount of specimen required to infect 50% of human embryonic fibrobrast cells [50% tissue culture infective dose (TCID50)] was determined. In preliminary experiments, we found that changing the gamma -globulin-free calf serum (GIBCO BRL) (19, 32) to ultra-low IgG FCS did not affect RV titration.

Viral infection of human tracheal epithelial cells. Medium was removed from confluent monolayers of human tracheal epithelial cells and replaced with 1 ml of DMEM-Ham's F-12 medium containing 2% USG. RV 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 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). 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 at either 1, 6, 12, or 24 h after RV infection. Furthermore, to measure the viral release for either 1-3, 3-5, or 5-7 days, 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 24 h after RV infection. The whole volume of the medium was taken for measurement of the viral content, and the same volume of fresh medium was replaced on days 3 and 5. The whole volume of the medium was taken for measurement of the viral content on day 7. The supernatants were stored at -80°C for determination of the viral content. Cell-associated viral content was also analyzed with sonicated human tracheal epithelial cells in MEM. The 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 an antibody to the LDL receptor on RV infection. Confluent human tracheal epithelial cells were incubated for 30 min at 37°C with medium alone, with medium containing a mouse anti-human monoclonal antibody to the LDL receptor (50 µg/ml; Oncogene Research Products, Cambridge, MA), or with medium containing an isotype-matched mouse IgG2b, kappa  control monoclonal antibody (50 µg/ml; PharMingen, San Diego, CA). The monoclonal antibody to the LDL receptor is the IgG2b, kappa  isotype, and it recognizes an epitope of the ligand binding region of the LDL receptor (5). After excess antibody was washed off, the monolayers were exposed to RV2 (105 TCID50/ml) or RV14 (105 TCID50/ml) for either 15 or 60 min before the monolayers were rinsed, and fresh 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 was added. The viral content of this medium was then assessed at various times after infection.

Detection of RV RNA by RT-PCR. Human tracheal epithelial cells cultured in glass tubes were lysed by the addition of RNAzol (0.2 ml/106 cells; BIOTECX, Houston, TX) and 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.

PCR was performed as previously described (12, 32). Briefly, 2 µg of RNA from each aliquot of human tracheal epithelial cells were dissolved in 100 µl of a buffer containing the reagents with the following composition for the RT reaction: 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 5 U/µl of Moloney murine leukemia virus RT (GIBCO BRL), 0.5 mM deoxynucleotide 5'-triphosphate (Takara, Ohtsu, Japan), 1 U/µl of 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 10 min at 95°C. The resulting cDNA was frozen at -80°C until used in the PCR. For each sample, 5 µl of the RT mixture were added to 45 µl of a PCR mixture consisting of 10 mM Tris · HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide 5'-triphosphate, and 1.25 U of Taq polymerase (Takara). 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 (32). 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 an ultraviolet illuminator and photographed with type 667 positive/negative film (Polaroid, Cambridge, MA).

For the RVs, RNA expression in the human tracheal epithelial cells was examined before and 8 h and 1, 3, and 5 days after RV2 infection.

Cytokine assays. Because RV infection increased the production of various cytokines from primary cultures of human tracheal epithelial cells (32), we measured interleukin (IL)-1alpha , IL-1beta , IL-6, IL-8, tumor necrosis factor (TNF)-alpha , granulocyte-macrophage colony-stimulating factor (GM-CSF), 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 ELISA kit (Ohtsuka, Tokushima, Japan), the IL-1beta ELISA kit (Ohtsuka), 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); 3 pg/ml for the IFN-gamma ELISA kit (Genzyme, Cambridge, MA); 1 U/ml for the IFN-beta ELISA kit (BioSource International, Camarillo, CA); and 2 pg/ml for the GM-CSF ELISA kit (Genzyme). 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.

To determine the effects of an antibody to the LDL receptor on the production of cytokines induced by RV2 and RV14, confluent human tracheal epithelial cells were incubated for 30 min with medium alone or with a medium containing either a monoclonal antibody to the LDL receptor (50 µg/ml) (5) or an isotype-matched mouse IgG2b, kappa  control monoclonal antibody (50 µg/ml) at 37°C before RV2 or RV14 infection.

Northern blot analysis. Northern blot analysis was done as previously described (27, 32). 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 Science). The LDL receptor cDNA probe (2.8 kb) was prepared by digesting the plasmid pLDLr3 (American Type Culture Collection, Manassas, VA) with the restriction endonucleases HindIII and SmaI (26). The membrane was hybridized with [alpha -32P]dCTP (3,000 Ci/mmol; Amersham)-labeled human LDL receptor cDNA 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-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. Quantification of the autoradiographic bands was accomplished with an image analyzer (Bio Imaging Analyzer, BAS-2000; Fuji Photo Film). We used an average value from replicate cultures from the same trachea (n = 3) for analysis of the intensity of the LDL receptor or beta -actin bands.

To study the effects of RV infection on the mRNA expression of the LDL receptor in human tracheal epithelial cells, cells were examined 8 h and 1, 3, and 5 days after RV2 infection and 1 day after RV14 infection.

To determine the mechanisms responsible for the upregulation of LDL receptor mRNA expression after RV2 infection, we tested the effects of either IL-1beta (200 pg/ml; Genzyme), IL-6 (100 pg/ml; Genzyme), IL-8 (100 pg/ml; Collaborative Research, Bedford, MA), or TNF-alpha (10 pg/ml; Genzyme) on LDL receptor mRNA expression in human tracheal epithelial cells. The concentration of each cytokine chosen was matched to a net increase in the culture medium after RV2 infection. Cells were incubated overnight with each cytokine.

Flow cytometry analysis of cell membrane LDL receptor expression. Induction of LDL receptor expression in human tracheal epithelial cells after infection with either RV2 (105 TCID50/ml for 60 min) or RV14 (105 TCID50/ml for 60 min) was assayed by flow cytometry analysis as previously described (30). Cells in glass tubes were collected by incubation with 0.02% EDTA for 10 min at 37°C, and a volume containing 1.5 × 106 cells was centrifuged for 7 min at 425 g, resuspended in ice-cold PBS-0.5% BSA, and washed twice with ice-cold PBS-0.5% BSA for 5 min at 541 g. The final pellet was resuspended in 300 µl of ice-cold PBS-0.5% BSA and divided into aliquots for 2 samples and 1 control. Then, 12.5 µl of an anti-LDL receptor antibody solution (0.5 µg/µl; Oncogene Research Products) were added to each sample and incubated for 30 min at 4°C. Subsequently, the samples were washed twice with ice-cold PBS-0.5% BSA and incubated with 100 µl of FITC-labeled goat-anti-mouse IgG (DAKO quantification kit QIFIKIT, DAKO, Glostrup, Denmark) for 45 min at 4°C. The samples were then washed twice with ice-cold PBS-0.5% BSA and incubated with 5 µl of phycoerythrin-labeled mouse anti-human CD14 IgG (DAKO). The cells were washed twice more and resuspended in 500 µl of PBS-0.5 BSA-0.1% sodium azide. Controls were processed in parallel with the samples but received an equivalent volume of PBS-0.5% BSA-0.1% sodium azide instead of primary antibody. In addition, 100 µl of Set-Up bead suspension (QIFIKIT) and 100 µl of Calibration bead suspension (QIFIKIT) were processed in parallel but received equivalent volumes of PBS-0.5% BSA-0.1% sodium azide instead of the anti-LDL antibody and anti-CD14 antibody. All measurements were performed on a flow cytometer (FACSCalibur, Becton Dickinson). Subsequently, the resultant population was reviewed in a forward-scatter versus side-scatter window, and only the core of the population was used for subsequent LDL receptor determination in the FL1 (FITC) channel. The acquisition number of the final population was set at 10,000. Standardized equivalents of LDL receptor density per cell, expressed as sites per cell (S/C units), were calculated by mathematical transformation of mean fluorescence intensities into specific antibody binding capacities with a calibration curve derived from the Calibration beads (QIFIKIT, DAKO). The resultant specific antibody binding capacity represents an equivalent of LDL receptor expression (30).

Effects of neutralizing antibodies to IL-1beta and TNF-alpha on RV2 infection and LDL receptor mRNA expression. To determine the role of endogenous IL-1beta in viral infection and LDL receptor expression, confluent human tracheal epithelial cells were preincubated with a mouse anti-human IL-1beta monoclonal antibody (10 µg/ml; Genzyme) or an isotype-matched mouse IgG1 control monoclonal antibody (Chemicon International) at the same concentration for 5 days (32). We also tested the effects of a mouse anti-human TNF-alpha monoclonal antibody (10 µg/ml, 5 days; Genzyme) on viral infection and LDL receptor expression. Viral titers in the supernatants collected for 3-5 days and the expression of LDL receptor mRNA 5 days after RV2 infection (105 TCID50/ml) were measured in confluent human tracheal epithelial cells preincubated with each antibody.

Effect of IL-1beta on susceptibility to RV2 infection. To examine whether IL-1beta increases the susceptibility to either RV2 or RV14 infection, confluent human tracheal epithelial cells were preincubated with and without IL-1beta (200 pg/ml) for 24 h. The epithelial cells were then exposed to serial twofold dilutions of either RV2 or RV14 (103 TCID50/ml) for 1 h at 33°C before being rinsed, and 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 was added. The presence of RV2 or RV14 in 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 RV2 and RV14 used. This index of susceptibility to infection, defined as the minimum dose of RV that could induce infection, was compared with the susceptibility of control cells that were not incubated with IL-1beta (31).

Isolation of nuclear extracts. Nuclear extracts were prepared with the methods previously described (10, 39). To isolate the nuclear extracts, all procedures were performed on ice. The human tracheal epithelial cells were washed with ice-cold calcium- and magnesium-free PBS, harvested by scraping into calcium- and magnesium-free PBS, and pelleted in a 1.5-ml microfuge tube at 1,850 g for 5 min. After these procedures were repeated once more, 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 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 the debris by centrifugation, the protein concentration of the nuclear extract was determined. The nuclear extracts were then stored at -80°C until used.

Electrophoretic mobility shift assay. Electrophoretic mobility shift assays (EMSAs) were performed as previously described (8, 10, 22, 39). The radiolabeled double-strand oligonucleotide probe for either the nuclear factor-kappa B (NF-kappa B) or SP1 site was prepared by annealing complementary oligonucleotides and by end labeling with [gamma -32P]ATP and T4 polynucleotide kinase. The radiolabeled probes used for NF-kappa B and SP1 were composed of the following sequences: 5'-AGTTGAGGGGACTTTCCCAGGC-3' for NF-kappa B (7, 20) and 5'-AATTACCGGGCGGGCGGGCTACCGGGCGGGCT-3' for SP1 (Stratagene, La Jolla, CA) (10). 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 the 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 RV2-induced NF-kappa B-DNA binding (39). 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. Preimmune serum (Santa Cruz Biotechnology) was used to control for any nonspecific effects of these antisera on SP1 activation (10).

Statistical analysis. Results are expressed as means ± SE. Statistical analysis was performed with two-way repeated-measures ANOVA. Subsequent post hoc analysis was made with Bonferroni's method. Significance was accepted at P < 0.05; n is the number of donors from which cultured epithelial cells were used.


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RV infection of human tracheal epithelial cells. Exposing confluent human tracheal epithelial cell monolayers to RV2 and RV14 (105 TCID50/ml) consistently led to infection. Collection of culture medium at different times after viral exposure revealed no detectable virus 1 h after infection. Both RV2 and RV14 were detected 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 RV2 or RV14 (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 RV2 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, 2.3 ± 0.3 log TCID50 units at 24 h, 2.6 ± 0.3 log TCID50 units at 3 days, 2.1 ± 0.3 log TCID50 units at 5 days, and 1.5 ± 0.3 log TCID50 units at 7 days (n = 7 each). 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 ± 0.3 log TCID50 units at 5 days, and 1.7 ± 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 24 h after RV2 infection in cells from smokers did not differ significantly from those in nonsmokers (3.1 ± 0.3 log TCID50 units in smokers vs. 3.2 ± 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 RV2-infected cultures. Likewise, RV2 infection did not alter the amount of lactate dehydrogenase in the supernatants (32 ± 3 IU/l before vs. 35 ± 3 IU/l 5 days after infection; P > 0.20; n = 7). RV2 infection also had no effect on cell number. Cell counts 24 h after infection were not significantly different (1.8 × 106 ± 0.2 × 106 in noninfected cells vs. 1.8 × 106 ± 0.3 × 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 the 105 units of rhinovirus (RV) required to infect 50% of human embryonic fibroblast cells [50% tissue culture infective dose (TCID50)]/ml. A: viral titers of RV2 (open circle ) and RV14 () in supernatants collected at sequential times during the 1st 24 h after infection. B: viral titers of RV2 (open bars) and RV14 (solid bars) in supernatants collected for indicated times after infection. Results are means ± SE from 7 samples.

Detection of viral RNA by PCR. Further evidence of RV2 infection in human tracheal epithelial cells and of viral replication was provided by PCR analysis (Fig. 2). In each of three experiments, RNA extracted from control uninfected cells did not produce any detectable PCR product at 385 bp (0 h). A faint product band was observable in RNA extracted from cells 8 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 RV RNA from human tracheal epithelial cells after RV2 infection as detected by RT-PCR. beta -Actin was used as a housekeeping gene. M, Phi X174/HincII fragment molecular weight marker; -, absence; +, presence. Data are representative of 3 different experiments.

Effects of RV infection on cytokine production. 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 RV2 (A) or RV14 (B) infection. Because viral infection did not alter cell number (see RV infection of human tracheal epithelial cells), all cytokine values are reported in picograms per milliliter of supernatant. Basal secretion was quite high with IL-8 and 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 RV2 and RV14, although in terms of absolute levels, IL-1beta (204 ± 18 pg/ml in RV2 and 157 ± 13 pg/ml in RV14) and IL-6 (182 ± 19 pg/ml in RV2 and 127 ± 13 pg/ml in RV14) predominated. RV2 infection did not alter the production of IL-1alpha (15 ± 2 pg/ml 3 days after RV2 infection vs. 14 ± 2 pg/ml 3 days after sham infection; P > 0.20; n = 7) and GM-CSF (152 ± 11 pg/ml 3 days after RV2 infection vs. 167 ± 21 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 RV2 and sham infections throughout the experiments.


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Fig. 3.   Time course of release of cytokines into supernatants of human tracheal epithelial cells after RV2 (A) and RV14 (B) infection (). open circle , Sham infection (control). IL, interleukin; TNF-alpha , tumor necrosis factor-alpha . Results are means ± SE from 7 samples. Significant difference from corresponding control value: * P < 0.05; ** P < 0.01.

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

Effects of an antibody to the LDL receptor on RV infection and cytokine production. Incubation of cells with a monoclonal antibody to the LDL receptor completely blocked RV2 infection as assessed by the absence of detectable viral titers in the supernatants recovered 24 h after 15 min of RV2 exposure (2.1 ± 0.2 log TCID50 units in control cells and 0 ± 0 log TCID50 units with the LDL receptor antibody). Likewise, a monoclonal antibody to the LDL receptor significantly decreased viral titers in the supernatants (3.3 ± 0.2 log TCID50 units in control cells vs. 2.1 ± 0.2 log TCID50 units with the LDL receptor antibody; P < 0.01; n = 7) as well as those in cell lysates (2.3 ± 0.2 log TCID50 units in control cells vs. 1.1 ± 0.1 log TCID50 units with the LDL receptor antibody; P < 0.01; n = 7) 24 h after 60 min of RV2 exposure. Treatment with the LDL receptor antibody also significantly inhibited increases in IL-1beta , IL-6, IL-8, and TNF-alpha production induced by RV2 infection (Fig. 4A). However, an isotype-matched IgG2b, kappa  control monoclonal antibody failed to alter viral titers in the supernatants 24 h after 15 min of RV2 exposure (2.2 ± 0.2 log TCID50 units; P > 0.50; n = 7) and 60 min of RV2 exposure (3.4 ± 0.3 log TCID50 units; P > 0.50; n = 7). Likewise, IgG2b, kappa  control monoclonal antibody did not inhibit increases in IL-1beta , IL-6, IL-8, and TNF-alpha production induced by RV2 (Fig. 4A). In contrast to RV2, viral titers in the supernatants recovered 24 h after 15 min of RV14 exposure were not altered by a monoclonal antibody to the LDL receptor (2.1 ± 0.2 log TCID50 units; P > 0.50; n = 7) from the control value (2.2 ± 0.1 log TCID50 units; n = 7) and from the value of the IgG2b, kappa  control monoclonal antibody treatment (2.1 ± 0.1 log TCID50 units; n = 7). Likewise, viral titers in cell lysates 24 h after 60 min of RV14 exposure were not altered by a monoclonal antibody to the LDL receptor (2.1 ± 0.1 log TCID50 units; P > 0.50; n = 7) from the control value (2.2 ± 0.2 log TCID50 units; n = 7). Neither a monoclonal antibody to the LDL receptor nor IgG2b, kappa  control monoclonal antibody altered increases in the production of IL-1beta , IL-6, IL-8, and TNF-alpha induced by RV14 infection (Fig. 4B).


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Fig. 4.   Release of cytokines into supernatants of human tracheal epithelial cells in the presence of a monoclonal antibody to the low-density lipoprotein (LDL) receptor (solid bars) or a mouse purified IgG2b, kappa  monoclonal antibody (hatched bars) or in the absence of any antibody (control; open bars). Effects of a monoclonal antibody to the LDL receptor and a mouse IgG2b, kappa  monoclonal antibody were examined at the maximal production of each cytokine after RV2 (A) and RV14 (B) infection. Results are means ± SE from 7 samples. Significant difference from viral infection alone: * P < 0.05; ** P < 0.01.

Effect of RV infection on LDL receptor expression. The baseline expression of LDL receptor mRNA was constant for at least 5 days in confluent human tracheal epithelial cell sheets, and the coefficient of variation was small (9.7%; n = 22). Neither smoking habit nor cause of death influenced the baseline expression of LDL receptor mRNA. Exposure of human tracheal epithelial cells to RV2 (Fig. 5A) or RV14 (Fig. 5B) caused increases in LDL receptor mRNA compared with a sham exposure (control, 0 h). Human tracheal epithelial cells 24 h after RV2 (Fig. 5C) or RV14 (Fig. 5D) infection were shown to overexpress LDL receptor mRNA fourfold compared with those 24 h after a sham exposure. IL-1beta (200 pg/ml) also increased LDL receptor mRNA (0.35 ± 0.03 scan units; P < 0.01; n = 7). However, neither IL-6 (200 pg/ml), IL-8 (100 pg/ml), nor TNF-alpha (10 pg/ml) altered LDL receptor mRNA levels (0.24 ± 0.02 scan units with IL-6; 0.23 ± 0.03 scan units with IL-8, and 0.21 ± 0.02 scan units with TNF-alpha ; P > 0.20; n = 7) compared with those after sham exposure (0.22 ± 0.02 scan units; n = 7).


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Fig. 5.   Northern blot analysis demonstrating increases in LDL receptor (LDL-R) mRNA levels in human tracheal epithelial cells at indicated times after RV2 (A) and RV14 (B) infection compared with those after sham infection (0 h; control). beta -Actin was used as a housekeeping gene. C and D: expression of LDL receptor mRNA in human tracheal epithelial cells 24 h after RV2 and RV14 infection, respectively. LDL receptor mRNA was normalized to a constitutive expression of beta -actin mRNA. Results are means ± SE from 7 samples. Significant difference from corresponding control value, ** P < 0.01.

Expression of the LDL receptor was also assayed by flow cytometry analysis. Human tracheal epithelial cells 3 and 5 days after RV2 infection were shown to increase the LDL receptor-specific fluorescence intensity compared with those after a sham exposure (Fig. 6). Human tracheal epithelial cells 5 days after RV14 infection were also shown to increase the LDL receptor-specific fluorescence intensity compared with those after a sham exposure (10.0 × 104 ± 0.7 × 104 S/C units 5 days after RV14 infection vs. 6.5 × 104 ± 0.6 × 104 S/C units 5 days after sham infection; P < 0.01; n = 7). IL-1beta (200 pg/ml, 24 h) also increased the LDL receptor-specific fluorescence intensity (8.5 × 104 ± 0.6 × 104 S/C units in IL-1beta vs. 6.5 × 104 ± 0.6 × 104 S/C units in control cells; P < 0.05; n = 7).


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Fig. 6.   Flow cytometry analysis demonstrating increases in LDL receptor expression in human tracheal epithelial cells 5 days after RV2 infection (A) compared with that after sham infection (B). C: LDL receptor-specific fluorescence intensity in human tracheal epithelial cells before (day 0) and 1, 3, and 5 days after infection RV2 (). open circle , Sham infection (control). s/c, Sites/cell. Results are mean ± SE from 7 samples. Significant difference from sham infection: * P < 0.05; ** P < 0.01.

Effects of neutralizing antibodies to IL-1beta and TNF-alpha on viral infection and LDL receptor expression. The mouse anti-human IL-1beta monoclonal antibody (10 µg/ml) caused significant decreases in RV2 titers in the supernatants collected on days 3-5 (2.8 ± 0.3 log TCID50 units/ml; P < 0.01; n = 7) compared with those in the control supernatants (4.0 ± 0.3 log TCID50 units/ml; n = 7) as well as decreases in LDL receptor mRNA expression 5 days after RV2 infection (0.62 ± 0.04 scan units with IL-1beta antibody vs. 0.78 ± 0.03 scan units in control cells; P < 0.05; n = 7) in human tracheal epithelial cells. In contrast, neither the mouse anti-human TNF-alpha monoclonal antibody (10 µg/ml) nor the mouse IgG1 control monoclonal antibody (10 µg/ml) altered viral titers of the supernatants (4.0 ± 0.3 log TCID50 units/ml with anti-human TNF-alpha antibody and 4.1 ± 0.2 log TCID50 units/ml with IgG1 control antibody; P > 0.50; n = 7) and LDL receptor mRNA expression (0.78 ± 0.03 scan units with anti-human TNF-alpha antibody and 0.79 ± 0.02 scan units with IgG1 control antibody; P > 0.50; n = 7).

The mouse anti-human IL-1beta monoclonal antibody (10 µg/ml) also caused a significant reduction in LDL receptor protein expression in human tracheal epithelial cells (7.8 × 104 ± 0.6 × 104 S/C units; P < 0.05; n = 7) compared with that 5 days after RV2 infection alone (11.0 × 104 ± 0.7 × 104 S/C units; n = 7). In contrast, neither the mouse anti-human TNF-alpha monoclonal antibody (10 µg/ml) nor the mouse IgG1 control monoclonal antibody (10 µg/ml) altered LDL receptor protein expression (11.3 × 104 ± 0.7 × 104 S/C units with anti-human TNF-alpha antibody and 11.0 × 104 ± 0.8 × 104 S/C units with IgG1 control antibody; P > 0.50; n = 7).

Effect of IL-1beta on the susceptibility to RV2 infection. Pretreatment of human tracheal epithelial cells for 24 h with IL-1beta (200 pg/ml) increased the susceptibility of cells to RV2 infection, decreasing by twofold the minimum dose of virus necessary to cause infection (1.5 ± 0.1 log TCID50 units with IL-1beta vs. 2.1 ± 0.1 log TCID50 units in control cells; P < 0.05; n = 7).

NF-kappa B and SP1 DNA-binding activity in human tracheal epithelial cells. Nuclear extracts from the human tracheal epithelial cells with RV2 or sham infection contained activated SP1 and NF-kappa B as demonstrated by the presence of a complex consisting of protein bound to a DNA fragment carrying the NF-kappa B site (Fig. 7A) and SP1 (Fig. 7B). The baseline intensity of NF-kappa B and SP1 DNA-binding activity was constant, and increased activation of NF-kappa B and SP1 was present in cells from 0.5 h and continued for up to 12 h after RV2 infection (Fig. 7). The activation of NF-kappa B and SP1 then decreased with longer incubations. 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. 8). The 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 during cell culture. However, the supershifting of the NF-kappa B band was not observed with antibody to either p52, c-Rel, or Rel B or preimmune antiserum (Fig. 8). The band showing SP1 DNA-binding activity was completely abolished by an exogenous addition of a 50-fold excess of an unlabeled DNA fragment (data not shown).


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Fig. 7.   Electrophoretic mobility shift assay (EMSA) demonstrating increases in nuclear factor (NF)-kappa B (A) and SP1 (B) DNA-binding activities (arrows) of human tracheal epithelial cells before (0) and at indicated times after RV2 infection.



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Fig. 8.   Identification of RV2-induced NF-kappa B bands with supershift 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, RV2-induced NF-kappa B DNA-binding activities; arrowhead, supershifted bands caused by antisera against p50 and p65.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that minor group RV2 can infect primary cultures of human tracheal epithelial cells via binding to the LDL receptor on the epithelial cells as shown previously in human fibroblasts (11) and HeLa cells (16) and upregulates the production of proinflammatory cytokines and the LDL receptor in epithelial cells. These conclusions are based on the observation that RV2 titers of culture supernatants and lysates from infected cells and RV2 RNA from infected cells increased with time. RV2 infection increased the production of IL-1beta , IL-6, IL-8, and TNF-alpha in supernatants and upregulated the expression of mRNA and protein of the LDL receptor in the cultured human tracheal epithelial cells. Furthermore, an antibody to the LDL receptor reduced RV2 titers in the culture supernatants and lysates from infected cells and RV2 infection-induced cytokine production in the culture supernatants.

Viral infection of cultured human tracheal epithelial cells and subsequent viral replication were confirmed by showing the increased viral content in the culture supernatants of infected cells with time as assessed by the cytopathic effects of this culture supernatant 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 with 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 RV2 were consistently observed when confluent monolayers were exposed to this virus. However, RV2 infection failed to influence both cell number and cell viability. This is in agreement with previous studies (31, 32, 37) showing the lack of cytotoxicity on epithelial cells in RV infection.

The specificity of the infection process for primary cultures of human tracheal epithelial cells by RV2 was confirmed by demonstrating that infection could be blocked with an antibody to the LDL receptor but not by an isotype-matched IgG2b, kappa  monoclonal antibody. Furthermore, a monoclonal antibody to the LDL receptor failed to block RV14 infection, a major group RV that uses ICAM-1 as its receptor (9, 33). 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 (25). The potency of inhibition by a monoclonal antibody to the LDL receptor observed in the present study is consistent with that in the previous study (16) in which RV2 binding was reduced to about one-third when RV2 was present during the incubation of the cells with a soluble LDL receptor fragment. In addition, we showed that the antibody to the LDL receptor inhibited the production of IL-1beta , IL-6, IL-8, and TNF-alpha induced by RV2 infection without an effect on RV14-induced increases in cytokine production. However, an antibody to the LDL receptor could not achieve complete inhibition of cytokine production induced by RV2 infection, which may also be due to the longer incubation times described above.

We showed that infection with either RV2 or RV14 as well as IL-1beta at the experimentally measured concentration in supernatants upregulated LDL receptor expression as assessed by increases in LDL receptor mRNA with Northern blot analysis and LDL protein with flow cytometry analysis. IL-1beta increased the susceptibility to RV2 infection by only twofold despite increases in susceptibility to RV14 infection by >10-fold (32), suggesting that an endogenous IL-1beta may upregulate LDL receptor expression less than ICAM-1 expression in the cultured human tracheal epithelial cells. These findings are consistent with previous reports (10, 13, 24) that IL-1 and TNF-alpha increase ICAM-1 expression by >10-fold in pulmonary epithelial cells, but they increase LDL receptor mRNA expression by only 5- and 6-fold in hepatocellular carcinoma cells and vascular endothelial cells. The anti-IL-1beta antibody significantly inhibited RV2 infection and RV2 infection-induced increases in mRNA expression and protein of the LDL receptor. However, the inhibitory effects of the anti-IL-1beta antibody on LDL receptor expression and RV2 infection were smaller than those on ICAM-1 expression and RV14 infection (32). Therefore, the role of endogenous IL-1beta in LDL receptor expression and RV2 infection may be minor. Other factors regulating RV2 infection need to be clarified in the future.

Increased activation of NF-kappa B was present in cells from 0.5 h after RV2 infection in the present study. The time course of NF-kappa B activation is consistent with previous reports (23, 39) in airway epithelial cells caused by infection with RV14 or RV16. NF-kappa B increases the expression of genes for many cytokines, enzymes, and adhesion molecules including ICAM-1 (4, 23, 39). Therefore, RV2-induced cytokine production may be associated with the activation of NF-kappa B in human tracheal epithelial cells. In contrast, another transcription factor, SP1, but not NF-kappa B, is reported to mediate the TNF-alpha -induced LDL receptor expression and sterol-induced repression of LDL receptor expression (7, 10, 15). We observed that RV2 infection also induces activation of SP1 in human tracheal epithelial cells. Therefore, LDL receptor expression by RV2 infection may be mediated, in part, through activation of SP1. A variety of intracellular signals such as several kinases and intracellular calcium are suggested to activate NF-kappa B (3) and SP1 (21). Because stimulation of the LDL receptor with LDL induces protein kinase C activation in smooth muscle cells (28), protein kinase C may, in part, relate to the activation of NF-kappa B and SP1 in airway epithelial cells, although we did not examine the relationship between the LDL receptor and the intracellular signals. Further studies are needed to clarify the mechanisms.

RV infection induces local production of cytokines, known to mediate the acute-phase reactions of airway inflammation (31, 32, 39). The species of cytokine and time course of cytokine production differ between cell types. For example, RV14 infection increases production of IL-6, IL-8, and GM-CSF in the airway epithelial cell line BEAS-2B (31) and that of IL-6 in A549 alveolar epithelial type II-like cells (39). In the present study, RV2 induced the production of IL-1beta , IL-6, IL-8, and TNF-alpha in human tracheal epithelial cells, which is consistent with a previous report (32). Although an increase in TNF-alpha production after infection was subtle and in IL-8 was <30% from the baseline, there was a large amount of IL-1beta production from human tracheal epithelial cells, 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 production, prostaglandin E2 synthesis, and degranulation from neutrophils (25). IL-1beta also causes increases in ICAM-1 expression on both epithelial and vascular endothelial cells (2, 6, 34). Upregulation of ICAM-1 on HeLa cells (33) and human tracheal epithelial cells (32) is shown to be associated with the increased binding of major group RVs.

Both RV2 and RV14 infections increased the expression of mRNA and protein of the LDL receptor in the human tracheal epithelial cells. The physiological and pathological role of the LDL receptor expression in the airway epithelial cells is still uncertain. However, upregulation of the LDL receptor on human fibroblasts is associated with increased binding of RV2 (11), suggesting an increase in susceptibility to minor group RV infection. Therefore, both major and minor subgroup RVs may cause a predisposition to other serotypes of minor group RV infection through increasing the expression of its receptor, the LDL receptor. RV 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 that infection of minor group RV2 induces LDL receptor expression. RV2 infection upregulated the production of cytokines that regulate the acute-phase reaction of airway inflammation. Furthermore, an antibody to the LDL receptor inhibited RV2 infection and the production of cytokines in response to RV2 infection. These findings suggest that minor group RV2 can be infectious through binding to the LDL receptor in primary cultures of human tracheal epithelial cells. Because major group RV14 also induced LDL receptor expression, both major and minor RVs may amplify their own and the other group's RV infection by overexpression of the LDL receptor and ICAM-1 (23, 32) on epithelial cells. These processes may be relevant to airway inflammation and exacerbations of asthma induced by minor group RVs.


    ACKNOWLEDGEMENTS

We thank Grant Crittenden for the English correction and Akira Ohmi, Michiko Okamoto, 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 1-1, Aoba-ku, 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 4 July 2000; accepted in final form 16 October 2000.


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