Department of Geriatric Medicine, Tohoku University School of Medicine, Sendai 980; and Virus Center, Clinical Research Division, Sendai National Hospital, Sendai 983, Japan
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ABSTRACT |
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Exacerbations of
asthma are often associated with respiratory infection caused by
rhinoviruses. To study the effects of rhinovirus infection on
respiratory epithelium, a primary target for respiratory viruses, human
rhinovirus (HRV)-2 and HRV-14 were infected to primary cultures of
human tracheal epithelial cells. Viral infection was confirmed by
showing that viral titers of supernatants and lysates from infected
cells increased with time and by polymerase chain reaction. HRV-2 and
HRV-14 infections upregulated the expression of intercellular adhesion
molecule-1 (ICAM-1) mRNA, the major rhinovirus receptor, on epithelial
cells, and they increased the production of interleukin (IL)-1,
IL-6, IL-8, and tumor necrosis factor (TNF)-
in supernatants.
Antibodies to ICAM-1 inhibited HRV-14 infection of epithelial cells and
decreased the production of cytokines after HRV-14 infection, but they
did not alter HRV-2 infection-induced production of cytokines. IL-1
upregulated ICAM-1 mRNA expression and increased susceptibility to
HRV-14 infection, whereas other cytokines failed to alter ICAM-1 mRNA
expression. Furthermore, a neutralizing antibody to IL-1
significantly decreased viral titers of supernatants and ICAM-1 mRNA
expression after HRV-14 infection, but a neutralizing antibody to
TNF-
was without effect. Immunohistochemical studies revealed that
both HRV-14 infection and IL-1
increased ICAM-1 expression on
cultured epithelial cells. These findings imply that HRV-14 infection
upregulated ICAM-1 expression on epithelial cells through increased
production of IL-1
, thereby increasing susceptibility to infection.
These events may be important for amplification of airway inflammation after viral infection in asthma.
asthma; common cold; airway inflammation; interleukin-1; intercellular adhesion molecule-1; polymerase chain reaction
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INTRODUCTION |
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PERSPECTIVE STUDIES have indicated that asthma attacks are associated with a viral infection in as many as 20-50% of the cases (17). Rhinoviruses cause the majority of common colds, which often provoke wheezing in patients with asthma (18), and they have also been associated with exacerbations of chronic bronchitis (11). Although several mechanisms have been proposed, it is still uncertain how viral respiratory infections cause an attack of wheezing in patients with asthma.
Respiratory tract epithelium is the primary target for respiratory
viruses. Respiratory syncytial viruses cause an increase in interleukin
(IL)-8 mRNA expression of the nasal epithelium (4) and the production
of IL-6, IL-8, and granulocyte macrophage colony-stimulating factor
(GM-CSF) from a human bronchial epithelial cell line (BEAS-2B; see Ref.
19). Likewise, human rhinovirus (HRV) infection induces production of
IL-6, IL-8, and GM-CSF from BEAS-2B cells (27). These cytokines are
known to mediate a wide variety of proinflammatory and immunoregulatory
effects (1) and to play a role in the pathogenesis of airway
inflammation in asthma. Subauste et al. (27) demonstrated that
preexposure of BEAS-2B cells to tumor necrosis factor- (TNF-
)
increased susceptibility to HRV-14 infection. However, HRV-14 infection itself failed to increase susceptibility to infection and intercellular adhesion molecule-1 (ICAM-1) expression, a major surface receptor for
rhinoviruses (7). We speculate that a lack of increased susceptibility
to infection may be due to the use of the BEAS-2B cell line in the
study by Subauste et al. (27). The BEAS-2B cell was derived from
normal human epithelial cells by transfection with an adenovirus
12-SV40 hybrid virus (25). Although BEAS-2B cells share many properties
in common with normal epithelial cells (27), transformed cell lines may
lose certain differentiated functions (8), resulting in an altered
response of respiratory epithelial cells to rhinovirus infection.
We therefore investigated whether the primary cultures of the human tracheal epithelial cells (34) can be infected with rhinoviruses. We also examined whether rhinovirus infection upregulates ICAM-1 expression on the cells and increases susceptibility to infection.
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METHODS |
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Media components.
Reagents for cell culture media were obtained as follows: Eagle's
minimum essential medium (MEM), Dulbecco's modified Eagle's medium
(DMEM), Ham's F-12 medium, fetal calf serum (FCS), and -globulin-free calf serum (GGFCS) were from GIBCO-BRL Life
Technologies, Palo Alto, CA; trypsin, EDTA, dithiothreitol, Sigma type
XIV protease, human placental collagen, penicillin, streptomycin,
gentamicin, and amphotericin B were from Sigma Chemical, St. Louis, MO;
and Ultroser G serum substitute (USG) was from BioSepra, Marlborough, MA.
Human embryonic fibroblast cell culture. Human embryonic fibroblast cells were cultured in the Roux type bottle (Iwaki Garasu, Chiba, Japan) sealed with a rubber plug in MEM containing 10% FCS supplemented with 5 × 104 U/l penicillin and 50 mg/l streptomycin (20). Confluency was achieved at 7 days, at which time the cells were collected by trypsinization (0.05% trypsin, 0.02% EDTA). Cells (1.5 × 105 cells/ml) suspended in MEM containing 10% FCS were then plated in glass tubes (15 × 105 mm; Iwaki Garasu), sealed with rubber plugs, and cultured at 37°C.
Human tracheal epithelial cell culture. Tracheas for cell culture were obtained 3-6 h after death from 51 patients (mean age, 64 ± 4 yr; 22 female, 29 male) under a protocol passed by the Tohoku University Ethics Committee. Twenty-four of the patients were smokers. None had a respiratory illness, and they died of acute myocardial infarction (n = 13), congestive heart failure (n = 3), malignant tumor other than lung cancer (n = 14), rupture of aortic aneurysm (n = 4), liver cirrhosis (n = 3), renal failure (n = 3), leukemia (n = 3), malignant lymphoma (n = 1), cerebral bleeding (n = 6), and cerebral infarction (n = 1). Tracheas were rinsed in ice-cold and sterile phosphate-buffered saline (PBS) to remove mucus and debris, opened longitudinally along the anterior surface, and mounted in a stretched position in a dissection tray. The surface epithelium was scored into longitudinal strips and was pulled off of the submucosa (34). The tissue strips were rinsed four times in PBS containing 5 mM dithiothreitol and then two times in PBS alone. The tissue strips were placed into the conical tubes (Costar, Cambridge, MA) containing protease (0.4 mg/ml; Sigma type XIV) dissolved in PBS. The strips were stored overnight in the refrigerator at 4°C. The enzyme was then competitively inhibited by the addition of FCS to a final concentration of 2.5%, and small sheets of cells were dislodged from the epithelial strips by vigorous agitation. The denuded strips were removed, and the sheets of cells remaining were dispersed by repeated aspiration in a 10-ml pipette.
Cells were pelleted (200 g, 10 min) and suspended in DMEM-Ham's F-12 containing 5% FCS (50:50 vol/vol). Cell counts were made using a hemocytometer, and estimates of viability were done using trypan blue and by measuring the amount of lactate dehydrogenase (LDH) in the medium as previously reported (2). The cells were then plated at 5 × 105 viable cells/ml in glass tubes coated with human placental collagen (34). This medium was replaced by DMEM-Ham's F-12 containing 2% USG on the first day after plating. The glass tubes were sealed with rubber plugs and were cultured at 37°C. Cell culture media were supplemented with 105 U/l penicillin, 100 mg/l streptomycin, 50 mg/l gentamicin, and 2.5 mg/l amphotericin B. We screened 16 kinds of viruses (e.g., influenza types A, B, and C, parainfluenza virus, adenovirus, rhinovirus, and respiratory syncytial virus) in supernatants of cultured human tracheal epithelial cells before rhinovirus infection using the method described previously (20) and used the epithelial cell sheets without contamination by any viruses. Furthermore, we confirmed cilia beating on the epithelial cells and the absence of fibroblasts in glass tubes using the inverted microscope (MIT-2; Olympus, Tokyo, Japan). Finally, to determine whether cultured cells can form tight junctions, we performed parallel cultures of human tracheal epithelial cells on Millicell CM inserts (0.45-µm pore size and 0.6-cm2 area; Millipore Products Division, Bedford, MA) to measure electrical resistance and short-circuit current using Ussing chamber methods (34). When cells cultured under these conditions become differentiated and form tight junctions without contamination of fibroblasts, they have values of >40Viral stocks.
HRV-2 and HRV-14 were prepared in our laboratory from the patients with
common colds (20). Stocks of HRV-14 and HRV-2 were generated by
infecting human embryonic fibroblast cells cultured in glass tubes in 1 ml of MEM supplemented with 2% GGFCS, 50 U/ml penicillin, and 50 µg/ml streptomycin at 33°C. The cells were incubated for several
days in glass tubes in 1 ml of MEM supplemented with 2% GGFCS until
cytopathic effects were obvious, after which the cultures were frozen
at 80°C, thawed, and sonicated. The virus-containing fluid so
obtained was frozen in aliquots at
80°C. The content of viral
stock solutions was determined using the human embryonic fibroblast
cell assay described below.
Detection and titration of viruses. Rhinoviruses were detected by exposing confluent human embryonic fibroblast cells in glass tubes to serial 10-fold dilutions of virus-containing medium or lysates in MEM supplemented with 2% GGFCS. Glass tubes were then incubated at 33°C for 7 days, and the cytopathic effects of viruses on human embryonic fibroblast cells were observed using an inverted microscope (MIT; Olympus) as reported previously (20). The amount of specimen required to infect 50% of human embryonic fibroblast cells (TCID50) was determined.
Viral infection of human tracheal epithelial cells.
Medium was removed from confluent monolayers of human tracheal
epithelial cells and was replaced with 1 ml of DMEM-Ham's F-12 containing 2% USG. Rhinovirus was added at a concentration of 105 TCID50/ml. After a 1-h incubation
at 33°C, the viral solution was removed, and the cells were rinsed
one time with 1 ml of PBS. The cells were then fed with DMEM-Ham's
F-12 containing 2% USG supplemented with 105 U/l
penicillin, 100 mg/l streptomycin, 50 mg/l gentamicin, and 2.5 mg/l
amphotericin B and were cultured at 33°C with rolling in the
incubator (HDR-6-T; Hirasawa, Tokyo, Japan). Supernatants were removed
at various times after infection and were stored at 80°C for the
determination of viral content. Viral titers of the material used for
infection and of the supernatants removed at the end of the infection
period were also determined to estimate the maximal amount of viral
uptake that had occurred during the exposure period. As an additional
control for nonspecific adherence of virus, the content of inocula
after a 1-h incubation in empty glass tubes was also assessed. Finally,
cell-associated viral content was analyzed using sonicated human
tracheal epithelial cells. Viral content in the supernatant and
cell-associated viral content are expressed as TCID50 units
per milliliter and as TCID50 units per 106
cells, respectively.
Effects of antibodies to ICAM-1 on rhinovirus infection. Confluent human tracheal epithelial cells were incubated for 30 min at 37°C with medium alone, with medium containing either of the two mouse monoclonal anti-human antibodies to ICAM-1 [84H10 (100 µg/ml; Immunotech, Marseille, France) or RR1 (100 µg/ml; a gift from Boehringer Ingelheim, Ridgefield, CT; see Ref. 14)], or with medium containing an isotype-matched mouse immunoglobulin G1 (IgG1) control monoclonal antibody (100 µg/ml; Chemicon International). Both 84H10 and RR1 are IgG1 isotypes and recognize the ICAM-1 functional domain. After excess antibodies were washed off, the monolayers were exposed to HRV-2 (105 TCID50/ml) or HRV-14 (105 TCID50/ml) for either 15 or 60 min before rinsing and adding fresh DMEM-Ham's F-12 containing 2% USG supplemented with 105 U/l penicillin, 100 mg/l streptomycin, 50 mg/l gentamicin, and 2.5 mg/l amphotericin B. The viral content of this medium was then assessed at various times after infection.
Detection of rhinovirus RNA and cytokine mRNA by reverse
transcription-polymerase chain reaction.
Human tracheal epithelial cells cultured in the glass tubes were lysed
by the addition of RNazol (0.2 ml/106 cells; BIOTECX,
Houston, TX) and were transferred into Eppendorf tubes. The cell
homogenates were mixed with a 10% volume of chloroform, shaken
vigorously for 15 s, placed on ice for 15 min, and centrifuged at
12,000 g for 15 min at 4°C. The upper aqueous phase
containing RNA was collected and mixed with an equal volume of
isopropanol. Pellets of RNA were obtained by centrifugation at 12,000 g for 15 min at 4°C, dissolved in a water, and stored at
80°C before use.
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Cytokine assays.
In the preliminary study, we found that the mRNA of IL-1, IL-6,
IL-8, TNF-
, and GM-CSF were expressed in cultured human tracheal
epithelial cells before and after HRV-14 infection. To determine the
effects of HRV-2 or HRV-14 infection on the production of cytokines, we
measured the amount of IL-1
, IL-6, IL-8, and TNF-
released from
human tracheal epithelial cells into the culture medium before and at
1, 3, and 5 days after HRV-2 or HRV-14 infection. We also measured the
amount of GM-CSF and IL-1
in the supernatants of human tracheal
epithelial cells before and after HRV-14 infection and the amount of
IL-1
and TNF-
in the viral stocks. Proteins of IL-1
, IL-1
,
IL-6, IL-8, TNF-
, and GM-CSF were measured by specific enzyme-linked
immunosorbent assays (ELISA). Sensitivities of the assays were 10 pg/ml
for the IL-1
and IL-1
ELISA kit (Ohtsuka, Tokushima, Japan), the
IL-6 ELISA kit (Toray, Tokyo, Japan), and the IL-8 ELISA kit (Toray), 4 pg/ml for the TNF-
ELISA kit (Ohtsuka), and 2 pg/ml for the GM-CSF
ELISA kit (Genzyme, Cambridge, MA). We used an average value of
replicate cultures from the same trachea (n = 3) for the
analysis of cytokine production.
Northern blot analysis.
Northern blot analysis was done as described previously (26). Equal
amounts of total RNA (10 µg) extracted from human tracheal epithelial
cells, as determined spectrophotometrically, were subjected to
electrophoresis in 1% agarose-formaldehyde gel. The gel was then
transferred via capillary action onto a nylon membrane
(Hybond-N+; Amersham Life Science). The membrane was
hybridized with [-32P]dCTP (3,000 Ci/mmol;
Amersham)-labeled human ICAM-1 cDNA (1.8-kb Xba I fragment;
British Bio-technology, Oxon, UK) with a random-prime labeling kit
(Random Primer; Takara). Hybridization with a radiolabeled probe was
performed overnight at 42°C. After high-stringency washing was
performed (1× standard saline citrate, 0.1% sodium dodecyl sulfate,
60°C), autoradiographic detection of the hybridized probe was
performed by exposing Kodak Scientific Imaging film for 48-72 h at
70°C. Quantitation of autoradiographic bands was accomplished with
an image analyzer (Bio Imaging Analyzer, BAS-2000; Fuji Photo Film) and
was expressed as the intensity of the ICAM-1/
-actin bands. We used
an average value of replicate cultures from the same trachea
(n = 3) for analysis of the intensity of the ICAM-1/
-actin bands.
Effects of neutralizing antibodies to IL-1 and
TNF-
on HRV-14 infection and ICAM-1 mRNA expression.
To determine the role of endogenous IL-1
in viral infection and
ICAM-1 expression, confluent human tracheal epithelial cells were
preincubated using a monoclonal mouse anti-human IL-1
(10 µg/ml;
Genzyme) or an isotype-matched mouse IgG1 control
monoclonal antibody (Chemicon International), at the same concentration
for 5 days. We also tested the effects of a monoclonal mouse anti-human TNF-
(10 µg/ml, 5 days; Genzyme) on viral infection and ICAM-1 expression. Viral titers in the supernatants collected during 3-5
days and the expression of ICAM-1 mRNA 5 days after HRV-14 infection
(105 TCID50/ml) were measured in
confluent human tracheal epithelial cells preincubated with each
antibody.
Effects of IL-1 on susceptibility to HRV-14
infection.
To examine whether IL-1
increases the susceptibility to HRV-14
infection, confluent human tracheal epithelial cells were preincubated
with or without IL-1
(200 pg/ml) for 24 h. The epithelial cells were
then exposed to serial 10-fold dilutions of HRV-14 for 1 h at 33°C.
The presence of HRV-14 in supernatants collected during 1-3 days
after infection was determined using the human embryonic fibroblast
cell assay described above to assess whether infection occurred at each
dose of HRV-14 used. This index of susceptibility to infection, defined
as the minimum dose of HRV-14 that could induce infection, was compared
with the susceptibility of control cells that were not preincubated
with IL-1
(27).
Immunohistochemical analysis.
Immunohistochemical analysis for ICAM-1 expression in human tracheal
epithelial cells was done as described previously (21). Human tracheal
epithelial cells were cultured on Vitrogen gels on Millicell inserts
(0.45 µm pore size and 0.6 cm2 area; Millipore Products
Division; see Ref. 34). Cell sheets were fixed in
periodate-lysine-paraformaldehyde at 4°C for 2 h. After a wash in
sucrose (10, 15, or 20%)-PBS, they were embedded in optimum cutting
temperature compound (Miles Laboratories, Naperville, IL) in liquid
nitrogen and were stored at 70°C until use. The staining was
performed using an alkaline phosphatase-anti-alkaline phosphatase
(APAAP) method. Cryostat sections (6 µm) were incubated with 75%
heat-inactivated normal human AB serum (Sigma) in
Tris-buffered saline (pH 7.6) containing 0.05 M Tris base, 0.15 M NaCl,
0.01 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, and 0.01% saponin (TBS[H + S]; Sigma) for 1 h at room
temperature and then with 20% normal rabbit serum (Dako) in
TBS[H + S] for 20 min at room temperature to block nonspecific
binding of the first and second antibodies. The sections were overlaid
with 4 µg/ml of the antibody 84H10 (Immunotech) or with an
isotype-matched mouse monoclonal antibody with the irrelevant
specificity (Chemicon International) in TBS[H + S] containing 1%
bovine serum albumin (TBS[H + S]-BSA, fraction V; Sigma). After
overnight incubation at 4°C, the sections were incubated with
anti-mouse immunoglobulin rabbit immunoglobulins (Dako) diluted 40 times with TBS[H + S]-BSA for 30 min at room temperature followed
by incubation with soluble complexes of alkaline phosphatase
and mouse monoclonal anti-alkaline phosphatase (Dako) diluted 60 times with TBS[H + S]-BSA for 30 min at room temperature. After one
more repeat of these procedures, the sections were developed by
exposure to substrate for 8 min with the fast red substrate system
(Dako) according to the manufacturer's protocol and were
counterstained with hematoxylin.
Statistical analysis. Results are expressed as means ± SE. Statistical analysis was performed using a one-way analysis of variance (ANOVA), and multiple comparisons were made using Bonferroni's method. Significance was accepted at P < 0.05; n refers to the number of donors from which cultured epithelial cells were used.
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RESULTS |
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Rhinovirus infection of human tracheal epithelial cells. Exposing confluent human tracheal epithelial cell monolayers to HRV-2 or HRV-14 (105 TCID50/ml) consistently led to infection. Collection of culture medium at differing times after viral exposure revealed no detectable virus at 1 h after infection. Both HRV-2 and HRV-14 were detected in culture medium 6 h after infection, and the viral content progressively increased between 6 and 24 h after infection (Fig. 1A). Evidence of continuous viral production was obtained by the demonstration that the viral titers of supernatants collected during 1-3 days, 3-5 days, and 5-7 days after infection each contained significant levels of HRV-2 or HRV-14 (Fig. 1B). Analysis of the levels of cell-associated virus (the virus detectable in sonicates of the human tracheal epithelial cells) followed a similar time course to that observed in the medium. The viral titers of cell-associated HRV-14 were 0.0 ± 0.0 log TCID50 units at 1 h, 0.1 ± 0.1 log TCID50 units at 6 h, 0.8 ± 0.1 log TCID50 units at 12 h, 2.2 ± 0.3 log TCID50 units at 24 h, 2.5 ± 0.3 log TCID50 units at 3 days, 2.2 ± 0.3 log TCID50 units at 5 days, and 1.6 ± 0.3 log TCID50 units at 7 days (n = 7 each). In both cell supernatants and lysates, viral titer levels increased significantly with time (P < 0.05 in each case by ANOVA). Viral titers at 24 h after HRV-14 infection in cells from smokers did not differ significantly from those in nonsmokers (3.2 ± 0.3 log TCID50 units in smokers vs. 3.3 ± 0.3 log TCID50 units in nonsmokers; P > 0.50). Human tracheal cell viability, as assessed by the exclusion of trypan blue, was consistently >96% in HRV-14-infected culture. Likewise, HRV-14 infection did not alter the amount of LDH in the supernatants (29 ± 3 IU/l before vs. 33 ± 3 IU/l 5 days after infection; P > 0.20, n = 7). HRV-14 infection also had no effect on cell numbers. Cell counts 24 h after infection were not significantly different (1.7 ± 0.1 × 106 in noninfected cells vs. 1.7 ± 0.2 × 106 in infected cells; P > 0.50, n = 7).
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Detection of viral RNA by PCR. Further evidence of HRV-14 infection of human tracheal epithelial cells and of viral replication was provided by PCR analysis (Fig. 2). In each of three experiments, RNA extracted from uninfected cells did not produce any detectable PCR product at 381 bp (0 h). A faint product was observable in RNA extracted from cells 6 h after infection followed by a progressive increase in viral RNA until 3 days after infection.
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Effects of rhinovirus infection on cytokine production.
Human tracheal epithelial cells were screened for mRNA expression of
various cytokines. PCR analysis revealed mRNA expression for IL-1,
IL-6, IL-8, TNF-
, and GM-CSF before and after cells were exposed to
HRV-14 (105 TCID50/ml). However, mRNA
for IL-4, IL-5, IL-10, and interferon-
were not detectable in human
tracheal epithelial cells before and after HRV-14 infection in all
seven experiments (data not shown). Figure
3 shows the time course of IL-1
, IL-6, IL-8, and TNF-
production
in supernatants of human tracheal epithelial cells after HRV-14 (Fig.
3A) or HRV-2 (Fig. 3B) infection. Because viral
infection did not alter cell numbers (see above), all cytokine values
are reported in picograms per milliliter of supernatant. Basal
secretion was quite high with IL-8, relatively high with IL-6, but low
or negligible with IL-1
and TNF-
. However, secretion of IL-1
,
IL-6, IL-8, and TNF-
all increased in response to both HRV-2 and
HRV-14, although, in terms of absolute levels, IL-1
(195 ± 15
pg/ml in HRV-2 and 145 ± 15 pg/ml in HRV-14) and IL-6 (185 ± 22 pg/ml in HRV-2 and 120 ± 14 pg/ml in HRV-14) predominated. Of the cytokines measured, GM-CSF and IL-1
levels were not changed by HRV-14 infection (140 ± 10 pg/ml before vs. 165 ± 18 pg/ml after in GM-CSF and 12 ± 2 pg/ml before vs. 13 ± 2 pg/ml after in IL-1
; P > 0.20, n = 7).
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Effects of antibodies to ICAM-1 on rhinovirus infection and cytokine
production.
Incubation of cells with both mouse monoclonal antibodies to ICAM-1
completely blocked HRV-14 infection, as assessed by the absence of
detectable viral titers in the supernatants recovered 24 h after 15 min
of HRV-14 exposure (2.2 ± 0.2 log TCID50 units in
control, 0 ± 0 log TCID50 units in 84H10, and 0 ± 0
log TCID50 units in RR1). Likewise, viral titers 24 h after
60 min of HRV-14 exposure were significantly decreased by 84H10
(1.7 ± 0.3 log TCID50 units; P < 0.01,
n = 7) and RR1 (1.9 ± 0.3 log TCID50 units; P < 0.01, n = 7) from control values
(3.3 ± 0.1 log TCID50 units; n = 7). These
treatments also significantly inhibited increases in IL-1, IL-6,
IL-8, and TNF-
production induced by HRV-14 infection (Fig.
4A). However, an isotype-matched
IgG1 control monoclonal antibody failed to alter viral
titers in the supernatants 24 h after 15 min of viral exposure
(2.3 ± 0.2 log TCID50 units; P > 0.50, n = 7) and 60 min of viral exposure (3.3 ± 0.2 log
TCID50 units; P > 0.50, n = 7).
Likewise, IgG1 control monoclonal antibody did not inhibit
increases in IL-1
, IL-6, IL-8, and TNF-
production induced by
HRV-14 infection (Fig. 4A). In contrast to HRV-14, viral
titers in the supernatants recovered 24 h after 15 min of HRV-2
exposure were not altered by 84H10 (2.4 ± 0.2 log
TCID50 units; P > 0.50, n = 7) and
RR1 (2.3 ± 0.2 log TCID50 units; P > 0.50, n = 7) from control values (2.3 ± 0.2 log
TCID50 units; n = 7) and from values of
IgG1 control monoclonal antibody treatment (2.2 ± 0.3
log TCID50 units; n = 7). Neither 84H10, RR1, nor
IgG1 control monoclonal antibody altered increases in
production of IL-1
, IL-6, IL-8, and TNF-
induced by HRV-2
infection (Fig. 4B).
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Effect of rhinovirus infection on ICAM-1 mRNA expression.
The baseline expression of ICAM-1 was constant in confluent human
tracheal epithelial cell sheets, and the coefficient of variation was
small (9.8%; n = 22). Neither smoking habit nor cause of
death influenced the baseline expression of ICAM-1 mRNA. Exposure of
human tracheal epithelial cells to HRV-14 (Fig.
5A) or HRV-2 (Fig. 5B)
caused increases in ICAM-1 mRNA compared with sham exposure (control).
Human tracheal epithelial cells 5 days after HRV-14 (Fig. 5C)
or HRV-2 (Fig. 5D) infection were shown to overexpress ICAM-1
mRNA twofold compared with those 5 days after a sham exposure. IL-1
(200 pg/ml) increased ICAM-1 mRNA to the levels similar to those
induced by rhinovirus infection expressed as the intensity of the
ICAM-1/
-actin bands (0.49 ± 0.03 scan units;
P < 0.01, n = 7). However, neither IL-6 (100 pg/ml), IL-8 (100 pg/ml), nor TNF-
(10 pg/ml) altered the levels
(0.23 ± 0.03 scan units in IL-6, 0.22 ± 0.03 scan units in
IL-8, and 0.21 ± 0.03 scan units in TNF-
; P > 0.20,
n = 7) compared with sham exposure (0.21 ± 0.02 scan
units; n = 7).
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Effects of neutralizing antibodies to IL-1 and
TNF-
on viral infection and ICAM-1 mRNA expression.
The monoclonal mouse anti-human IL-1
(10 µg/ml) significantly
decreased HRV-14 titers of supernatant collected during 3-5 days
(Fig. 6A) and inhibited ICAM-1
mRNA expression in human tracheal epithelial cells (Fig. 6B).
In contrast, neither the monoclonal mouse anti-human TNF-
(10 µg/ml) nor mouse IgG1 control monoclonal antibody (10 µg/ml) altered viral titers of supernatant (Fig. 6A) and
ICAM-1 mRNA expression (Fig. 6B).
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Effects of IL-1 on susceptibility to HRV-14
infection.
Pretreatment of the human tracheal epithelial cells for 24 h with
IL-1
(200 pg/ml) increased the susceptibility of cells to HRV-14
infection, decreasing by 10-fold the minimum dose of virus necessary to
cause infection (1.1 ± 0.1 log TCID50 units in IL-1
vs. 2.2 ± 0.2 log TCID50 units in control;
P < 0.01, n = 7).
Immunohistochemical analysis.
Figure 7 shows ICAM-1 expression in the human tracheal epithelial
cells, which was detected as a red color. HRV-14 infection (Fig.
7B) increased ICAM-1 expression
in both apical and basolateral sides compared with sham infection,
which caused staining only on the basolateral side (Fig. 7A).
Treatment with IL-1 at either 200 pg/nl (Fig. 7C) or 10 ng/ml (Fig. 7D) mimicked HRV-14 infection, with a
stronger staining observed in the latter. In contrast, treatment with
either IL-6, IL-8, or TNF-
failed to alter ICAM-1 expression in the
human tracheal epithelial cells (not shown).
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DISCUSSION |
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Primary cultures of human airway epithelial cells are thought to be important for characterizing viral infection in the airway and for advancing our knowledge of airway inflammation (8). Respiratory viral infection of primary cultures of human nasal epithelial cells were reported previously, but these cultures contained a significant number of fibroblasts (32), making it difficult to interpret the results obtained. We report rhinovirus infection of primary cultures of the human tracheal epithelial cells that do not contain other cell types such as fibroblasts (34). However, it should be noted here that this model system has significant differences from airway epithelial cells in vivo.
Viral infection of cultured human tracheal epithelial cells and subsequent viral replication were confirmed by showing the increased viral content in the culture medium of infected cells with time, assessed by the cytopathic effects of this medium on human embryonic fibroblast cells, and by showing that the cytopathic effects of human tracheal epithelial cell lysates also increased with time after infection. Viral replication was also detected by PCR of viral RNA after reverse transcription into DNA. A progressive increase in viral RNA, observed until 3 days after infection, was detected by a pronounced band on PCR compared with the absence of any signal in RNA extracted from uninfected cells. Infections of human tracheal epithelial cells with HRV-2 and HRV-14 were consistently observed when confluent monolayers were exposed to virus. However, HRV-14 infection failed to influence both cell numbers and cell viability. This is in agreement with previous studies showing the lack of cytotoxicity on epithelial cells in rhinovirus infection (27, 32).
Infection of the respiratory epithelium by viruses has been shown to
cause increased production of cytokines (19, 27). Subauste et al. (27)
demonstrated that infection of BEAS-2B cells with HRV-14 induced an
increased production of IL-6, IL-8, and GM-CSF. The present study is in
agreement with a previous study showing the enhanced production of IL-6
and IL-8 within 24 h after infection (27). However, infection of human
tracheal epithelial cells with HRV-2 and HRV-14 also caused increases
in the production of IL-1 and TNF-
, which differed significantly from HRV-14 infection of BEAS-2B cells (27). Although an increase in
TNF-
production after infection was subtle and that in IL-8 was
<30% from the baseline, there was a large amount of IL-1
production from human tracheal epithelial cells in the present study,
which was maximal at 3 days and was sustained up to 5 days after
infection. IL-1
is a potent inflammatory cytokine that induces
growth and differentiation of T and B lymphocytes, other cytokine
productions, prostaglandin E2 synthesis, and degranulation from neutrophils (1). IL-1
also causes increases in ICAM-1 expression on both epithelial and vascular endothelial cells (1, 5,
29). Increases in ICAM-1 expression on vascular endothelial cells
promote the adhesion of neutrophils, monocytes, and lymphocytes to
these cells (5). Likewise, upregulation of ICAM-1 on HeLa cells is
associated with the increased binding of major group rhinoviruses (28).
The specificity of the infection process for primary cultures of human
tracheal epithelial cells by HRV-14 was confirmed by demonstrating that
infection could be blocked using antibodies to ICAM-1 but not by an
isotype-matched IgG1 monoclonal antibody. Furthermore,
antibodies to ICAM-1 failed to block HRV-2 infection, a minor group of
rhinoviruses that do not use ICAM-1 as its receptor. However,
inhibition became less consistent at longer incubation times (e.g., 1 h), presumably because of the high affinity of the virus for its
receptor and of the requirement for very few viral particles to enter
the cell to induce infection (27). In addition, we showed that
antibodies to ICAM-1 significantly inhibited the production of IL-1,
IL-6, IL-8, and TNF-
induced by HRV-14 but that HRV-2-induced
effects on cytokine production were not altered by antibodies to
ICAM-1. However, antibodies to ICAM-1 could not achieve complete
inhibition of cytokine production induced by HRV-14 infection, which
may be also due to longer incubation times described above.
Furthermore, we showed that rhinovirus infection and IL-1
at the
experimentally measured concentration in supernatants upregulated
ICAM-1 expression assessed by increases in ICAM-1 mRNA using Northern
blot analysis, whereas an increase in ICAM-1 mRNA was two times the
control and relatively small compared with the study on respiratory
syncytial virus infection of human pulmonary type II-like epithelial
(A549) cells (22). Similar to ICAM-1 mRNA levels, however, both HRV-14
infection and IL-1
increased ICAM-1 expression on epithelial cells
as shown by immunohistochemical analysis, and IL-1
increased
susceptibility to HRV-14 infection. Furthermore, enhancement of ICAM-1
mRNA expression by HRV-14 infection was almost entirely blocked by the
antibody to IL-1
, and the anti-IL-1
antibody significantly
inhibited HRV-14 infection, confirming the role of the endogenous
IL-1
in viral infection and ICAM-1 expression in the present study. Although TNF-
is reported to upregulate the expression of ICAM-1 on
epithelial cells (27), the present study failed to show the expression.
Furthermore, the antibody to TNF-
did not alter HRV-14 infection or
ICAM-1 mRNA expression. Lack of TNF-
-induced effects on ICAM-1 mRNA
expression is probably due to a small amount of TNF-
production from
human tracheal epithelial cells in response to HRV-14 infection.
Although IL-1
is reported to be induced by A549 cells in response to
respiratory syncytial virus infection (22), HRV-14 did not alter
IL-1
production in the present study. The discrepancy may be
explained by differences in the species of virus and cultured cells.
Upregulation of ICAM-1 expression on epithelial cells and the production of cytokines from these cells in response to rhinovirus infection may be relevant to the pathogenesis of airway inflammation associated with colds and mechanisms of viral exacerbations of asthma. IL-6 induces antibody production in B cells and T cell activation and differentiation (1). IL-8 is a major chemoattractant for neutrophils and stimulates neutrophils to cause enzyme release and production of reactive oxygen metabolites (12). Neutrophils and lymphocytes are shown to be predominant cell types in the nasal mucosa during rhinovirus infection (13). Likewise, upregulation of ICAM-1 could increase susceptibility to major group rhinoviruses (7, 28) and could lead cells adjacent to infected cells to infection when viruses are released from the cells originally infected. Furthermore, chronic antigen challenge is shown to increase ICAM-1 expression on airway epithelium, which may be related to airway inflammation in asthma (31). Inflammatory conditions such as asthma, smoking, and ozone exposure in which ICAM-1 expression is increased on respiratory epithelial surfaces may cause a predisposition to rhinovirus infection through increasing expression of the major group of rhinovirus receptors. The rhinovirus infection would enhance airway inflammation by recruiting neutrophils and, potentially, other inflammatory cells, causing increased mediator release and exacerbation of the underlying reactive airway diseases.
In summary, we have shown, for the first time, that rhinovirus is
infectious in primary cultures of human tracheal epithelial cells.
Rhinovirus infection upregulates ICAM-1 expression on epithelial cells.
Rhinovirus infection induces an increased production of cytokines that
regulate the acute phase reaction of airway inflammation. Of these,
IL-1 is able to upregulate ICAM-1 expression and to increase the
susceptibility to HRV-14 infection, and the antibody to IL-1
inhibited both viral infection and ICAM-1 expression. Furthermore,
antibodies to ICAM-1 reduce the production of cytokines induced by
HRV-14 infection. These findings suggest that rhinoviruses per se
amplify their infection by the overexpression of ICAM-1 on epithelial
cells through the production of IL-1
, resulting in a further
increase in the production of inflammatory cytokines. A recent report
has also demonstrated a similar IL-1
-dependent autocrine mechanism
in respiratory syncytial virus infection of A549 cells (22). Thus these
processes may be relevant to airway inflammation induced by respiratory
viruses and viral exacerbations of asthma.
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ACKNOWLEDGEMENTS |
---|
We thank Grant Crittenden for English correction and Akira Ohmi, Michiko Okamoto, and Minako Tada for technical assistance.
![]() |
FOOTNOTES |
---|
Address for reprint requests: H. Sasaki, Dept. of Geriatric Medicine, Tohoku University School of Medicine, Aoba-ku Seiryo-machi 1-1, Sendai 980, Japan.
Received 16 September 1996; accepted in final form 24 June 1997.
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