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
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ABSTRACT |
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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-B (NF-
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
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INTRODUCTION |
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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.
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METHODS |
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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 >40Viral 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 -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
-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 -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, control monoclonal antibody (50 µg/ml; PharMingen,
San Diego, CA). The monoclonal antibody to the LDL receptor is the
IgG2b,
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.
Cytokine assays.
Because RV infection increased the production of various cytokines from
primary cultures of human tracheal epithelial cells (32),
we measured interleukin (IL)-1, IL-1
, IL-6, IL-8, tumor necrosis
factor (TNF)-
, granulocyte-macrophage colony-stimulating factor
(GM-CSF), interferon (IFN)-
, IFN-
, and IFN-
by specific enzyme-linked immunosorbent assays (ELISAs). Sensitivities of the
assays were 25 pg/ml for the IFN-
ELISA kit (COSMO BIO, Tokyo, Japan); 10 pg/ml for the IL-1
ELISA kit (Ohtsuka, Tokushima, Japan),
the IL-1
ELISA kit (Ohtsuka), the IL-6 ELISA kit (Toray, Tokyo,
Japan), and the IL-8 ELISA kit (Toray); 4 pg/ml for the TNF-
ELISA
kit (Ohtsuka); 3 pg/ml for the IFN-
ELISA kit (Genzyme, Cambridge,
MA); 1 U/ml for the IFN-
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-
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-
. After the culture medium was
freeze-dried, the pellet was dissolved in 200 µl of water, and the
concentration of TNF-
was measured. The value was normalized
according to the medium volume.
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 [-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
-actin bands.
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-1 and TNF-
on RV2
infection and LDL receptor mRNA expression.
To determine the role of endogenous IL-1
in viral infection and LDL
receptor expression, confluent human tracheal epithelial cells were
preincubated with a mouse anti-human IL-1
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-
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-1 on susceptibility to RV2 infection.
To examine whether IL-1
increases the susceptibility to either RV2
or RV14 infection, confluent human tracheal epithelial cells were
preincubated with and without IL-1
(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-1
(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-B (NF-
B) or SP1 site was prepared by annealing complementary
oligonucleotides and by end labeling with [
-32P]ATP
and T4 polynucleotide kinase. The radiolabeled probes used for NF-
B
and SP1 were composed of the following sequences:
5'-AGTTGAGGGGACTTTCCCAGGC-3' for NF-
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-B
family were involved in RV2-induced NF-
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-
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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RESULTS |
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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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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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Effects of RV infection on cytokine production.
Figure 3 shows the time course of
IL-1, IL-6, IL-8, and TNF-
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-1
and
TNF-
. However, secretion of IL-1
, IL-6, IL-8, and TNF-
all
increased in response to both RV2 and RV14, although in terms of
absolute levels, IL-1
(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-1
(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-
, IFN-
, and IFN-
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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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-1, IL-6, IL-8,
and TNF-
production induced by RV2 infection (Fig. 4A). However, an
isotype-matched IgG2b,
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,
control monoclonal antibody did not inhibit increases in IL-1
, IL-6,
IL-8, and TNF-
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,
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,
control monoclonal antibody altered increases
in the production of IL-1
, IL-6, IL-8, and TNF-
induced by RV14
infection (Fig. 4B).
|
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-1
(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-
(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-
; P > 0.20; n = 7) compared with those after sham exposure (0.22 ± 0.02 scan
units; n = 7).
|
|
Effects of neutralizing antibodies to IL-1 and TNF-
on viral
infection and LDL receptor expression.
The mouse anti-human IL-1
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-1
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-
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-
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-
antibody and 0.79 ± 0.02 scan units with IgG1 control
antibody; P > 0.50; n = 7).
Effect of IL-1 on the susceptibility to RV2 infection.
Pretreatment of human tracheal epithelial cells for 24 h with
IL-1
(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-1
vs. 2.1 ± 0.1 log TCID50 units in control
cells; P < 0.05; n = 7).
NF-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-
B as demonstrated by
the presence of a complex consisting of protein bound to a DNA fragment
carrying the NF-
B site (Fig.
7A) and SP1 (Fig.
7B). The baseline intensity of NF-
B and SP1 DNA-binding activity was constant, and increased activation of NF-
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-
B and
SP1 then decreased with longer incubations. Specificity of the NF-
B
binding was confirmed by supershift EMSA in which antibodies to the p50
or p65 subunit of NF-
B ablated NF-
B bands (Fig.
8). The supershifting of the NF-
B band
with the antibody to the p50 or p65 subunit of NF-
B was constantly
observed at any time during cell culture. However, the supershifting of
the NF-
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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DISCUSSION |
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---|
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-1, IL-6, IL-8, and TNF-
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, 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-1
, IL-6, IL-8, and
TNF-
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-1 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-1
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-1
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-
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-1
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-1
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-1
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-B was present in cells from 0.5 h
after RV2 infection in the present study. The time course of NF-
B
activation is consistent with previous reports (23, 39) in
airway epithelial cells caused by infection with RV14 or RV16. NF-
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-
B in human tracheal epithelial cells. In contrast,
another transcription factor, SP1, but not NF-
B, is reported to
mediate the TNF-
-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-
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-
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-1, IL-6, IL-8, and TNF-
in human
tracheal epithelial cells, which is consistent with a previous report
(32). Although an increase in TNF-
production after
infection was subtle and in IL-8 was <30% from the baseline, there
was a large amount of IL-1
production from human tracheal epithelial
cells, which was maximal at 3 days and was sustained up to 5 days after
infection. IL-1
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-1
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.
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ACKNOWLEDGEMENTS |
---|
We thank Grant Crittenden for the English correction and Akira Ohmi, Michiko Okamoto, and Fusako Chiba for technical assistance.
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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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