1 Department of Geriatric
Medicine and 2 Department of
Otorhinolaryngology, To further
understand the early biochemical events that occur in infected surface
epithelium, we developed for the first time a model in which a
respiratory submucosal gland cell population can be infected with
rhinovirus (RV). Viral infection was confirmed by demonstrating with
PCR that viral titers in supernatants and lysates from infected cells
increased with time. Infection by RV14 upregulated the expression of
intercellular adhesion molecule-1 (ICAM-1) mRNA, the major RV receptor,
on submucosal gland cells, and it increased production of interleukin
(IL)-1
intercellular adhesion molecule-1; asthma; common cold; airway
inflammation; interleukin-1; polymerase chain reaction
RHINOVIRUSES (RVs) are the major cause of the common
cold, the most common acute infectious illness in humans (10).
Furthermore, 80% of asthma exacerbations in school-aged children and
half of all asthma exacerbations in adults are associated with viral
upper respiratory infection, and the majority of viruses isolated are RVs (18, 23).
Intercellular adhesion molecule-1 (ICAM-1), the receptor for the major
group of RVs (13), is expressed by airway epithelial cells (26, 32).
Likewise, during colds, RV has been detected in a limited number of
shed nasal epithelial cells by indirect immunofluorescence (34) as well
as in nasal epithelial cells from biopsies of infected subjects by in
situ hybridization (3). These findings indicate that the nasal
epithelium is an important site of RV infection in humans. Furthermore,
the literature (9, 11, 16) suggests RV infection in the lower
respiratory tract in humans. In contrast to a variety of other
respiratory pathogens (e.g., influenza and adenovirus), cytotoxicity of
epithelial cells does not appear to play a major role in the
pathogenesis of RV infections (8, 10, 15). Instead, it is believed that
the manifestations of RV-induced pathogenesis are the result of
virus-induced mediators of inflammation (37).
The submucosal glands produce airway secretions 40 times more than
goblet cells (28). The airway submucosal glands may be the target of RV
infection because nasal discharge in the common cold contains both
watery and mucus secretions (6, 14). Nasal discharge in RV infection is
reduced by a parasympatholytic agent and an antihistamine, but the
inhibitory effects are not complete and an antihistamine agent failed
to improve nasal obstruction (6, 14). Therefore, RV infection itself
may also relate to airway submucosal edema and secretions of the
submucosal glands. Histological examination showed RV infection-induced
infiltration of leukocytes in the airway mucosa and submucosa (12).
Therefore, cells in the submucosa may be involved in RV infection and
its infection-induced inflammation. Infection of epithelial cells with
RV induces production of several cytokines such as interleukin (IL)-1,
IL-6, IL-8, tumor necrosis factor (TNF)- To further understand the cellular events involved in the pathogenesis
of RV respiratory tract infection, studies were undertaken to determine
whether human respiratory submucosal gland cells can be infected with
RV and whether infection of these cells with RV leads to increased
production of proinflammatory cytokines and increases in ICAM-1
expression in human submucosal gland cells.
Medium components. Reagents for cell
culture medium 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 Human embryonic fibroblast cell
culture. Human embryonic fibroblast cells were cultured
in MEM containing 10% FCS and supplemented with 5 × 104 U/l of penicillin and 50 mg/l
of streptomycin in Roux-type bottles (Iwaki Garasu, Chiba, Japan) that
were sealed with rubber plugs (25). Confluency was achieved at 7 days,
at which time the cells were collected by trypsinization (0.05%
trypsin and 0.02% EDTA). The cells (1.5 × 105 cells/ml) suspended in the MEM
containing 10% FCS were then plated in glass tubes (15 × 105 mm;
Iwaki Garasu) that were sealed with rubber plugs and cultured at
37°C.
Human tracheal submucosal gland cell
culture. Tracheae from 41 patients without overt
pulmonary disease (ages 30-81 yr, median 65 yr; 16 women and 25 men) were obtained 3-6 h after death under a protocol passed by
the Tohoku University (Sendai, Japan) Ethics Committee.
Culture methods have been described in detail elsewhere (36). In brief,
the tracheae were rinsed in ice-cold 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
pulled off the submucosa. Gland-rich submucosal tissue was dissected
from the cartilage and the adventitia, immersed in fresh PBS, and
minced with scissors. Tissue fragments were recovered by centrifugation
(200 g for 10 min) and placed in
Hanks' buffered salt solution containing 20 mM HEPES buffer (pH 7.4), crude collagenase type IV (500 U/ml), pancreatic porcine elastase (6 U/ml), hyaluronidase (200 U/ml), and deoxyribonuclease (10 U/ml). After
12-16 h of disaggregation in a trypsinizing flask on an orbital
shaker (240 rpm) at room temperature, disaggregated tissue was
decanted, recovered by centrifugation (200 g for 10 min), washed twice, and
suspended in a mixture of 40% Ham's F-12 medium, 40% DMEM, and 20%
FCS (F-12-DMEM-FCS), plated into two T25 tissue culture flasks
(Corning), and incubated at 37°C in 5%
CO2-95% air. The fragments of
submucosal tissue remaining in the trypsinizing flask were again
exposed to enzymatic digestion as above, and the second collection of
dispersed gland acini was combined with the suspension of unattached
acini from the two T25 flasks. The
combined acini from these two sources were spun down, suspended in
fresh F-12-DMEM-FCS, and replated in the two T25 flasks containing the attached
acini from the first plating. The following morning, F-12-DMEM-FCS was
replaced with F-12-DMEM with 0.1% USG and the following growth
factors: 10 µg/ml of insulin, 5 µg/ml of transferrin, 20 ng/ml of
triiodothyronine, 0.36 µg/ml of hydrocortisone, 7.5 µg/ml of
endothelial cell growth supplement, 25 ng/ml of epidermal growth
factor, 0.1 µM retinoic acid, and 20 ng/ml of cholera toxin.
It took 14-21 days to achieve confluency, at which time the cells
were collected by trypsinization (0.05% trypsin and 0.02% EDTA). The
cells were pelleted (200 g for 10 min)
and suspended in a F-12-DMEM-FCS. Cell counts were made with a
hemocytometer, and estimates of viability were done with trypan blue
and by measuring the amount of lactate dehydrogenase in the medium as
previously reported (32). The cells were then plated at 5 × 105 viable cells/ml in glass
tubes. This medium was replaced with F-12-DMEM supplemented with 0.1%
USG and growth factors on the first day after plating. The glass tubes
were sealed with rubber plugs and cultured at 37°C. The cell
culture medium was supplemented with
105 U/l of penicillin, 100 mg/l of
streptomycin, 50 mg/l of gentamicin, and 2.5 mg/l of amphotericin B. All culture vessels were coated with human placental collagen (20 µg/cm2) (36). The absence of
fibroblasts in the glass tubes was confirmed with an inverted
microscope (MIT-2, Olympus, Tokyo, Japan). Under an inverted
microscope, the cultured human tracheal submucosal gland 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
spindle-shaped fibroblasts as shown by Winther et al. (35) but not on
submucosal gland 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 submucosal
gland cells on Millicell CM inserts (0.45 µm pore size and 0.6 cm2 area; Millipore Products
Division, Bedford, MA) to measure electrical resistance and
short-circuit current with Ussing chamber methods (36). When cells
cultured under these conditions form tight junctions without
contamination by fibroblasts, they have values of >40
To determine whether cultured cells possess immunologic characteristics
of submucosal gland cells, the expression of serous and mucous gland
cell secretory antigen was examined by using immunocytochemical methods
as previously described (5, 30). The cells cultured on glass coverslips
were fixed with 4% paraformaldehyde and 0.5% Triton X-100 in 0.1 M
phosphate buffer for 15 min at room temperature and were then washed
with PBS. Immunocytochemistry with a monoclonal antibody directed
against mucous (A1F8) or serous (B1D8) gland cells of the original
tissues (30) was performed with a modification of a biotin-avidin
procedure (5). The specimens were counterstained with methyl green.
Viral stocks. RV14 was prepared in our
laboratory from patients with the common cold (25, 32). Stocks of RV14
were generated by infecting human embryonic fibroblast cells cultured
in glass tubes in 1 ml of MEM supplemented with 2% GGFCS, 50 U/ml of
penicillin, and 50 µg/ml of streptomycin at 33°C. The cells were
incubated for several days in glass tubes in 1 ml of MEM supplemented
with 2% GGFCS until the cytopathic effects were obvious, after which the cultures were frozen at 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 or lysates in MEM supplemented with 2% GGFCS. The glass tubes
were then incubated at 33°C for 7 days, and the cytopathic effects
of the viruses on human embryonic fibroblast cells were observed with
an inverted microscope (MIT, Olympus) as previously reported (25, 32). The amount of specimen required to infect 50% of human embryonic fibroblast cells [50% tissue culture infectious dose
(TCID50)] was determined.
Furthermore, to confirm that the cytopathic effects on human embryonic
fibroblast cells were caused by RV infection, RNA was extracted from
culture supernatants of human tracheal submucosal gland cells in each
subject and RV RNA was detected with the methods described in
Detection of RV RNA and cytokine mRNA by
RT-PCR.
Viral infection of human tracheal submucosal gland
cells. The medium was removed from confluent monolayers
of human tracheal submucosal gland cells and replaced with 1 ml of
F-12-DMEM supplemented with 0.1% USG and growth factors. RV was added
at a concentration of 105
TCID50 units/ml. After a 1-h
incubation at 33°C, the viral solution was removed, and the cells
were rinsed one time with 1 ml of PBS. The cells were then fed with
F-12-DMEM containing 0.1% USG and growth factors 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) (32). The supernatants were removed at various times
after infection and were stored at Effects of antibodies to ICAM-1 on RV
infection. Confluent human tracheal submucosal gland
cells were incubated for 30 min at 37°C with medium alone, with
medium containing either of two mouse monoclonal anti-human antibodies
to ICAM-1 [84H10 (100 µg/ml; Immunotech, Marseilles, France) or
RR1 (100 µg/ml; a gift from Boehringer Ingelheim, Ridgefield,
CT)], or with medium containing an isotype-matched mouse IgG1
control 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 RV14
(105
TCID50 units/ml) for 60 min before
being rinsed and fresh F-12-DMEM containing 0.1% USG and growth
factors supplemented with 105 U/l
of penicillin, 10 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 and cytokine mRNA by
RT-PCR. Human tracheal submucosal gland cells cultured
in glass tubes were lysed by the addition of RNAzol (0.2 ml/106 cells; BIOTECX, Houston,
TX) and were transferred to 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 PCR was performed as previously described (4, 19). Briefly, 2 µg of
RNA from each aliquot of human tracheal submucosal gland cells were
dissolved in 100 µl of buffer containing the following reagents for
the RT reaction: 50 mM Tris · HCl (pH 8.3), 75 mM
KCl, 3 mM MgCl2, 10 mM
dithiothreitol, 5 U/µl of Moloney murine leukemia virus RT (GIBCO
BRL), 0.5 mM deoxynucleoside 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 For the RV and each cytokine, mRNA expression in the human tracheal
submucosal gland cells and RV RNA content in the culture supernatants
were examined before and 1, 3, and 5 days after RV14 infection.
Cytokine assays. In the preliminary
study, we found that the mRNAs of IL-1 To determine the effects of antibodies to ICAM-1 on the production of
cytokines induced by RV14, confluent human tracheal submucosal gland
cells were incubated for 30 min with medium alone, with medium
containing either 84H10 (100 µg/ml) or RR1 (100 µg/ml) antibody, or
medium containing an isotype-matched mouse IgG1 control monoclonal
antibody (100 µg/ml; Chemicon International) at 37°C before RV14
infection. To confirm that increases in the cytokines induced by RV14
infection were due to the effects of RV14 infection and not a
contaminant present in the viral stock, the ability of UV-inactivated
virus to induce increases in the cytokines was also examined. UV
inactivation was performed as previously described (17).
Northern blot analysis. Northern blot
analysis was done as previously described (29). Equal amounts of total
RNA (10 µg) extracted from human tracheal submucosal gland cells, as
determined spectrophotometrically, were subjected to electrophoresis in
1% agarose-formaldehyde gels. The gel was then transfered via
capillary action onto a nylon membrane (Hybond-N+, Amersham Life
Science). The membrane was hybridized with
[ To study the effects of RV14 infection on mRNA expression of ICAM-1 in
human tracheal submucosal gland cells, the cells were examined 1, 3, and 5 days after RV14 infection.
To determine the mechanisms responsible for the upregulation of ICAM-1
mRNA expression after RV14 infection, we tested the effects of either
IL-1 Effects of neutralizing antibodies to IL-1 and
TNF- Effects of IL-1 on susceptibility to RV14
infection. To examine whether IL-1 Statistical analysis. Results are
expressed as means ± SE; n is the
number of donors from which cultured submucosal gland cells were used.
Statistical analysis was performed with a two-way repeated-measures
ANOVA. Bonferroni's test was used to estimate the level of
significance of differences between means. For all analyses, values of
P < 0.05 were assumed to be significant.
Immunocytochemistry. Figure
1 shows immunocytochemistry of confluent
monolayers of human tracheal submucosal gland cells. Nearly all cells
(>95%) reacted with the monoclonal antibody A1F8 directed against
mucous cells (Fig. 1B) and with the
monoclonal antibody B1D8 directed against serous cells (Fig.
1C), although there was cell-to-cell
variability in the staining. As negative controls, we stained the cells
with an isotype-matched mouse monoclonal antibody with an irrelevant
specificity and obtained no positive signals (Fig.
1A).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, IL-1
, IL-6, IL-8, tumor necrosis factor-
, and
granulocyte-macrophage colony-stimulating factor in supernatants.
Antibodies to ICAM-1 inhibited RV infection of submucosal gland cells
and decreased the production of cytokines after RV infection. Both
IL-1
and IL-1
upregulated ICAM-1 mRNA expression and increased
susceptibility to RV infection, whereas other cytokines failed to alter
ICAM-1 mRNA expression. Furthermore, neutralizing antibodies to IL-1
and IL-1
significantly decreased the viral titers in supernatants
and ICAM-1 mRNA expression after RV infection, but a neutralizing
antibody to tumor necrosis factor-
was without effect. These
findings suggest that respiratory submucosal gland cells play an
important role in the initial stages of inflammation and provide useful
insights into the pathogenesis of RV infection.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (31, 32). These cytokines are known
to mediate a wide variety of proinflammatory and immunoregulatory effects (1) and may play an important role in the pathogenesis of RV
infections. However, the role of submucosal gland cells in RV infection
has not been investigated.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globulin free calf serum
(GGFCS) were from GIBCO BRL (Life Technologies, Palo Alto, CA);
trypsin, EDTA, dithiothreitol, Sigma type XIV protease, collagenase
type IV, pancreatic porcine elastase, hyaluronidase, deoxyribonuclease,
human placental collagen, retinoic acid, cholera toxin, penicillin,
streptomycin, gentamicin, and amphotericin B were from Sigma (St.
Louis, MO); Ultroser G serum substitute (USG) was from BioSepra
(Marlborough, MA); and insulin, transferrin, epidermal growth factor,
endothelial cell growth supplement, hydrocortisone, and
triiodothyronine were from Collaborative Research (Bedford, MA).
· cm2 for
resistance and >2 µA/cm2 for
short-circuit current (36). Therefore, cultured human tracheal submucosal gland cells able to form tight junctions without
contamination by fibroblasts were used for the following experiments
when the cultured cells in the tubes had a cuboidal or round-shaped
appearance and the cells on the Millicell CM inserts had high
resistance (>40
· cm2) and
high short-circuit current (>2
µA/cm2).
80°C, thawed, and sonicated. The virus-containing fluid so obtained was frozen in aliquots at
80°C. The content of the viral stock solution was determined
with the human embryonic fibroblast cell assay described in
Detection and titration of
viruses.
80°C for the
determination of viral content. Cell-associated viral content was also
analyzed with sonicated human tracheal submucosal gland cells. The
viral content in the supernatant and the cell-associated viral content
are expressed as TCID50 units per
milliliter and TCID50 units per
106 cells, respectively.
80°C before use. To
extract RNA from the culture supernatants, 200 µl of the culture
supernatants were mixed with 800 µl of RNAzol by inverting gently. A
10% volume of chloroform was added to the mixture, 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, mixed with 20 µg of
glycogen (Boehringer Mannheim) as a carrier, and then mixed with an
equal volume of isopropanol. Pellets of RNA were obtained by
centrifugation at 12,000 g for 15 min
at 4°C and dissolved in water.
80°C until used
in the PCR. For each sample, 5 µl of RT mixture were added to a
45-µl PCR mixture consisting of 10 mM Tris · HCl
buffer (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 0.2 mM deoxynucleoside
5'-triphosphate, and 1.25 U of
Taq polymerase (Takara). Primer pairs
for the RV and cytokines were present at 2 ng/µl. Sequences of the
PCR primer pairs used in these experiments 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. The samples were separated on a 2% agarose gel (FMC BioProducts, Rockland, ME) and stained for 30 min in 1 µg/ml of ethidium bromide. The DNA bands were visualized on an
ultraviolet (UV) illuminator and were photographed with type 667 positive/negative film (Polaroid, Cambridge, MA).
, IL-1
, IL-6, IL-8,
TNF-
, and GM-CSF were expressed in cultured human tracheal
submucosal gland cells before and after RV14 infection. To determine
the effects of RV14 infection on the production of cytokines, we
measured the amount of IL-1
, IL-1
, IL-6, IL-8, TNF-
, and
GM-CSF released from human tracheal submucosal gland cells into the
culture medium before and at 1, 3, and 5 days after RV14 infection. The
proteins of IL-1
, IL-1
, IL-6, IL-8, TNF-
, and GM-CSF were
measured by specific enzyme-linked immunosorbent assays (ELISAs).
Sensitivities of the assay were 10 pg/ml for the IL-1
, IL-1
(both
from Ohtsuka, Tokushima, Japan), IL-6, and IL-8 (both from Toray,
Tokyo, Japan) ELISA kits, 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 donors) for the analysis of
cytokine production.
-32P]dCTP (3,000 Ci/mmol; Amersham)-labeled human ICAM-1 cDNA (1.8-kb Xba I fragment; British BioTechnology,
Oxon, UK) with a random-primer labeling kit (Random Primer, Takara).
Hybridization with a radiolabeled probe was performed overnight at
42°C. After high-stringency washing was performed (1×
saline-sodium citrate and 0.1% sodium dodecyl sulfate, 60°C),
autoradiographic detection of the hybridized probe was performed by
exposing Kodak scientific imaging film for 48-72 h at
70°C. Quantitation of autoradiographic bands was
accomplished with an image analyzer (BioImaging analyzer BAS-2000; Fuji
photo film) and was expressed as the intensity of the ICAM-1 band to that of the
-actin band. We used an average value of replicate cultures from the same trachea (n = 3 donors) for analysis of the intensity of the ICAM-1 band to that of the
-actin band.
(200 pg/ml; Genzyme), IL-1
(200 pg/ml; Ohtsuka), IL-6 (300 pg/ml; Genzyme), IL-8 (10 ng/ml; Collaborative Research), TNF-
(10 pg/ml; Genzyme), or GM-CSF (200 pg/ml; Genzyme) on ICAM-1 mRNA
expression in human tracheal submucosal gland cells. The experimentally
measured concentrations in the supernatants were used for each cytokine.
on RV14 infection and ICAM-1 mRNA
expression. To determine the role of endogenous
cytokines in viral infection and ICAM-1 expression, confluent human
tracheal submucosal gland cells were preincubated with a monoclonal
mouse anti-human IL-1
(10 µg/ml; Genzyme), an anti-human IL-1
(10 µg/ml; Genzyme), an anti-human TNF-
(10 µg/ml; Genzyme), or
an isotype-matched mouse IgG1 control (10 µg/ml; Chemicon
International) antibody for 5 days. Viral titers in the supernatants
collected for 3-5 days and the expression of ICAM-1 mRNA 5 days
after RV14 infection (105
TCID50 units/ml) were measured in
confluent human tracheal submucosal gland cells preincubated with each antibody.
and IL-1
increase the susceptibility to RV14 infection, confluent human tracheal
submucosal gland cells were preincubated with and without IL-1
(200 pg/ml) or IL-1
(200 pg/ml) for 24 h. The submucosal gland cells were
then exposed to serial 10-fold dilutions of RV14 for 60 min at
33°C. The presence of RV14 in the supernatants collected for
1-3 days after infection was determined with the human embryonic
fibroblast cell assay described in Detection and
titration of viruses to assess whether infection
occurred at each dose of RV14 used. This index of susceptibility to
infection, defined as the minimum dose of RV14 that could induce infection, was compared with the susceptibility of control
cells that were not preincubated with IL-1
or IL-1
(31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (107K):
[in a new window]
Fig. 1.
Immunocytochemistry of confluent monolayers of human tracheal
submucosal gland cells with monoclonal antibodies directed against
mucous (A1F8; B) and serous (B1D8;
C) cells and a negative control
treated with an isotype-matched mouse monoclonal antibody
(A). Magnification, ×100.
RV infection of human tracheal submucosal gland
cells. Exposing confluent human tracheal submucosal
gland cell monolayers to RV14 (105
TCID50 units/ml) consistently led
to infection. Collection of culture medium at differing times after
viral exposure revealed no detectable virus 1 h after infection. RV14
was detected in culture medium 8 h after infection, and the viral
content progressively increased between 8 and 24 h after infection
(Fig. 2). Evidence of continuous viral
production was obtained by demonstrating that the viral titers in
supernatants collected for 1-3, 3-5, and 5-7 days after
infection each contained significant levels of RV14 (Fig. 2). Analysis
of the levels of cell-associated virus (the virus detectable in
sonicates of the human tracheal submucosal gland cells) followed a
similar time course to that observed in the medium (Fig. 2). In both
cell supernatants and lysates, viral titer levels increased
significantly with time (P < 0.05 in
each case by ANOVA). Human tracheal submucosal gland cell viability as
assessed by the exclusion of trypan blue was consistently >96% in
RV14-infected cultures. Likewise, RV14 infection did not alter the
amount of lactate dehydrogenase in the supernatants (31 ± 3 IU/l
before vs. 34 ± 3 IU/l 5 days after infection;
P > 0.20; n = 7). RV14 infection also had no
effect on cell numbers. Cell counts 24 h after infection were not
significantly different (6.0 ± 0.4 × 105 in noninfected cells vs. 6.0 ± 0.3 × 105 in infected
cells; P > 0.50; n = 7).
|
Detection of viral RNA by PCR. Further
evidence of RV14 infection of human tracheal submucosal gland cells, of
viral replication, and of viral release into the supernatants was
provided by PCR analysis (Fig. 3). In each
of three experiments, RNA extracted from control uninfected cells did
not produce any detectable PCR product at 381 bp (0 h). A clear product
band was observed when RNA extracted from cells 8 h after the infection
period was used (Fig. 3A). Likewise,
in each of three experiments, RNA extracted from control uninfected
culture supernatants did not produce any detectable PCR product at 381 bp (0 h). A clear product band was observed when supernatants extracted
8 h after the infection period were used (Fig.
3B). The time course of the viral
titers in supernatants and lysates detected by the cytopathic effects
of viruses on human embryonic fibroblast cells was similar to that of
the intensity of the RV RNA product extracted from supernatants and
infected cells detected by PCR analysis.
|
Effect of RV infection on cytokine
production. Human tracheal submucosal gland cells were
screened for mRNA expression of various cytokines. PCR analysis
revealed mRNA expression for IL-1, IL-1
, IL-6, IL-8, TNF-
, and
GM-CSF before and after cells were exposed to RV14
(105
TCID50 units/ml). However, mRNAs
for IL-4, IL-5, IL-10, and interferon-
were not detectable in human
tracheal submucosal gland cells before and after RV14 infection in all
seven experiments (data not shown). Figure
4 shows the time course of IL-1
,
IL-1
, IL-6, IL-8, TNF-
, and GM-CSF production in supernatants
from human tracheal submucosal gland cells after RV14 infection.
Because viral infection did not alter cell numbers (see
RV infection of human tracheal submucosal gland
cells), all cytokine values are reported in picograms
per milliliter of supernatant. Basal secretion was quite high with IL-8
but low or negligible with IL-1
, IL-1
, IL-6, TNF-
, and GM-CSF.
However, secretion of IL-1
, IL-1
, IL-6, IL-8, TNF-
, and GM-CSF
all increased in response to RV14.
|
Effects of anti-ICAM-1 on RV infection and cytokine
production. Incubation of cells with both mouse
monoclonal antibodies to ICAM-1 completely blocked RV14 infection as
assessed by the absence of detectable viral titers in the supernatants
recovered 24 h after 60 min of viral exposure (4.1 ± 0.3 log
TCID50 units in control, 0 ± 0 log TCID50 units in 84H10-treated,
and 0 ± 0 log TCID50 units in
RR1-treated cells). These treatments also completely inhibited
increases in IL-1, IL-1
, IL- 6, IL-8, TNF-
, and GM-CSF
production induced by RV14 infection (Fig.
5). Neither viral titers in the
supernatants (4.2 ± 0.2 log
TCID50 units; P > 0.50;
n = 7) nor virally induced cytokine
production (Fig. 5) were altered by an isotype-matched
IgG1 control monoclonal antibody.
Likewise, exposure to UV-inactivated virus resulted in the production
of only 16.1 ± 4.1 pg/ml of IL-1
, 17.8 ± 3.9 pg/ml of
IL-1
, 15.9 ± 4.4 pg/ml of IL-6, 4,660 ± 961 pg/ml of IL-8,
3.6 ± 1.0 pg/ml of TNF-
, and 18.8 ± 5.1 pg/ml of GM-CSF. Thus UV inactivation failed to increase cytokine production compared with a sham infection (P > 0.20;
n = 7).
|
Effects of RV infection and cytokines on ICAM-1 mRNA
expression. Human tracheal submucosal gland cells 5 days after RV14 infection were shown to overexpress ICAM-1 mRNA
threefold compared with those 5 days after a sham exposure (Fig.
6). Both IL-1 (200 pg/ml; 0.38 ± 0.02 scan units; P < 0.01;
n = 7) and IL-1
(200 pg/ml; 0.35 ± 0.02 scan units; P < 0.01;
n = 7) significantly increased ICAM-1
mRNA levels compared with those after a sham exposure (0.19 ± 0.02 scan units; n = 7). However, neither
IL-6 (300 pg/ml), IL-8 (10 ng/ml), TNF-
(10 pg/ml), nor GM-CSF (200 pg/ml) altered the levels (0.20 ± 0.02 scan units for IL-6, 0.19 ± 0.02 scan units for IL- 8, 0.20 ± 0.02 scan units for
TNF-
, and 0.21 ± 0.03 scan units for GM-CSF;
P > 0.20;
n = 7).
|
Effects of neutralizing antibodies to cytokines on RV
infection and ICAM-1 mRNA expression. The combination
of monoclonal mouse anti-human IL-1 (10 µg/ml) and anti-human
IL-1
(10 µg/ml) antibodies significantly decreased RV14 titers in
supernatants collected for 3-5 days (Fig.
7A) and
significantly inhibited ICAM-1 mRNA expression in human tracheal
submucosal gland cells (Fig. 7B). In
contrast, neither the monoclonal mouse anti-human TNF-
(10 µg/ml)
nor the mouse IgG1 control monoclonal antibody (10 µg/ml) altered the
viral titers in the supernatant (Fig.
7A) and ICAM-1 mRNA expression (Fig.
7B).
|
Effects of cytokine pretreatment on susceptibility to
RV14 infection. Pretreatment of the human tracheal
submucosal gland cells for 24 h with IL-1 (200 pg/ml) and IL-1
(200 pg/ml) increased the susceptibility of cells to RV14 infection,
decreasing by 10-fold the minimum dose of virus necessary to cause
infection (1.1 ± 0.1 log
TCID50 units for IL-1
-treated
and 1.1 ± 0.1 log TCID50 units
for IL-1
-treated cells vs. 2.1 ± 0.2 log
TCID50 units for control cells;
P < 0.01;
n = 7).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To understand the cellular and molecular events involved in the host response to RVs, culture systems of human respiratory epithelial cells have been used (15, 31, 32, 37). These studies demonstrated that RV triggers the release of inflammatory mediators that play an important role in the pathogenesis of RV infections. To gain further insights into the early biochemical events that occur in infected epithelial cells, we developed for the first time a culture system in which human respiratory submucosal gland cells can be infected with RV.
We judged cultured human tracheal submucosal gland cells as cells able to form tight junctions without contamination by fibroblasts and used them for the experiments when the cultured cells in the tubes had a cuboidal or round-shaped appearance and the cells on Milicell CM inserts had high electrical resistance and high short-circuit current (36). The RV infection caused cytopathic effects on spindle-shaped fibroblasts but not on submucosal gland cells with a cuboidal or round-shaped appearance, which is consistent with previous observations on airway epithelial cells and fibroblasts (25, 31, 32, 35). Furthermore, >95% of passaged cells reacted with antibodies against gland cells (30, 36), although there was cell-to-cell variability in the staining. Therefore, cultured human tracheal submucosal glands may make confluent sheets of a heterogeneous collection of gland cells. However, significant contamination by fibroblasts is unlikely in the present study.
The primary target of RV in human infection is the nasal mucosa (3, 6, 14, 34). However, small-particle aerosols of RV have been shown to produce tracheobronchitis (9, 11). Cultures of sputum from children with wheezy bronchitis were more often positive for RV than nasal swabs taken at the same time (16). These findings suggest that RV infects the lower respiratory tract in humans.
Airway mucosal epithelial cells secrete RV into the mucosal and serosal
sides (26, 32), and RV infection affects the barrier function of airway
epithelium (26). Therefore, RVs released into the submucosa or RVs that
penetrate across the airway mucosa might be infected to submucosal
glands. The infected airway submucosal glands may release RV and
inflammatory cytokines into the submucosal space and upregulate ICAM-1
production, thereby resulting in the activation of lymphocytes and
monocytes to secrete interferon- (12) and in the accumulation of
lymphocytes and eosinophils in the airway (11). Histamine release from
human peripheral blood leukocytes is activated by infection of
respiratory viruses (7). Therefore, RV replication, cytokine release,
and ICAM-1 induction in submucosal glands may play a role in the
initial stages in inflammation.
Viral infection of cultured human tracheal submucosal gland cells and subsequent viral replication were confirmed by showing the increased viral content in the culture medium of infected cells with time as assessed by the cytopathic effects of this medium on human embryonic fibroblast cells and by showing that the cytopathic effects of human tracheal submucosal gland cell lysates also increased with time after infection. RV can be distinguished from enterovirus, which also produces "entero-like" cytopathic effects on human embryonic fibroblasts, by inactivation of RV after treatment with acid (25). Viral replication in the human tracheal submucosal gland cells and release of RV into the culture supernatants were also detected by PCR of viral RNA after RT into DNA. Experimental conditions clearly demonstrated that viral RNA increased 8 h after infection as indicated by a pronounced band on PCR compared with the absence of any signal immediately after infection. The lack of any PCR signal immediately after infection, together with the inability to detect virus with the human embryonic fibroblast assay in either the supernatants or lysates of infected cells until 8 h postinfection, implies that very little virus is initially taken up during the infection process. However, RV14 infection failed to influence both cell growth rate and cell viability. This is in agreement with previous studies (8, 15, 31, 32, 35) showing the lack of cytotoxicity on epithelial cells in RV infection. The lack of cytotoxicity in this model facilitates studies of virally induced biochemical changes in respiratory submucosal gland cells because the possibility that any such changes are occurring as a result of cell death can be ruled out.
The specifity of the infection process for human tracheal submucosal gland cells by RV14 was confirmed by demonstrating that infection could be blocked with antibodies directed against the functional binding site of ICAM-1, the major RV receptor, but not by an isotype-matched IgG1 monoclonal antibody. In addition, we showed that antibodies to ICAM-1 completely inhibited virally induced cytokine production, whereas both an isotype-matched IgG1 and a UV-inactivated virus failed to inhibit them, suggesting that cytokine production requires active RV infection.
In recent years, it has become apparent that respiratory epithelial
cells play a much more active role in regulating airway inflammation
and that the stimulation of biochemical pathways in infected epithelial
cells could induce a series of events that contribute to the
pathogenesis of viral infections. Respiratory syncytial viruses cause
an increase in IL-8 mRNA expression in the nasal epithelium (4) and
production of IL-6, IL-8, and GM-CSF from a human bronchial epithelial
cell line (BEAS-2B) (24). Likewise, RV infection induces production of
IL-6, IL-8, and GM-CSF from BEAS-2B cells (31) and of IL-1, IL-6,
IL-8, and TNF-
from primary cultures of human tracheal epithelial
cells (32). In addition to the enhanced production of IL-1
, IL-6,
IL-8, and TNF-
as previously observed in human tracheal epithelial
cells with RV14 infection (32), infection of human tracheal submucosal gland cells with RV14 also caused increases in IL-1
and GM-CSF production.
The cytokines produced from submucosal gland cells in response to viral stimulation have biological properties that are of interest with respect to the pathogenesis of colds and asthma. IL-8 is a potent chemoattractant for and activator of neutrophils (2) and also has chemotactic activity for lymphocytes (20), the two predominant cell types in the nasal mucosa during RV infections (21). IL-6 cannot only induce B-cell differentiation and antibody production but is also capable of stimulating T-cell activation (1). Likewise, GM-CSF can prime both neutrophils and eosinophils for enhanced activation to chemical stimuli (22). Increased epithelial expression of IL-8, IL-6, and GM-CSF has been reported in asthmatic airways, and, therefore, virally induced production of these cytokines may be relevant to viral exacerbations of asthma.
Infection of submucosal gland cells with RV14 was found to upregulate
the expression of ICAM-1 on these cells. It has been shown that
exposure of epithelial cells to cytokines such as IL-1, IL-1
, and
TNF-
can upregulate the expression of ICAM-1 on these cells (27, 31,
32). Furthermore, in HeLa cells, upregulation of ICAM-1 by these
cytokines is associated with increased binding of major group RVs (33).
In this study, virally induced enhancement of ICAM-1 expression was
mimicked by IL-1
and IL-1
but not by IL-6, IL-8, TNF-
, and
GM-CSF at the experimentally measured concentrations in the
supernatants. Furthermore, a combination of antibodies to IL-1
and
IL-1
almost entirely blocked increases in ICAM-1 expression induced
by RV14 infection and significantly inhibited RV14 infection. Finally,
both IL-1
and IL-1
increased susceptibility to infection with
RV14 infection, consistent with their ability to increase the
expression of ICAM-1. These results confirmed the role of endogenous
IL-1 in viral infection and ICAM-1 expression in submucosal gland
cells. In contrast to IL-1, TNF-
failed to alter both susceptibility
to RV14 infection and ICAM-1 expression and anti-TNF-
was without an
effect on virally induced enhancement of ICAM-1 expression. This is in
agreement with a previous study by Terajima et al. (32) in
primary cultures of human tracheal epithelial cells. Inflammatory
conditions such as asthma, smoking, and ozone exposure in which ICAM-1
expression is increased on submucosal gland cells may cause a
predisposition to RV infection through the increased expression of the
major group RV receptors. The 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 demonstrate that a pure population of human respiratory submucosal gland cells can be infected with RV. Infection of submucosal gland cells with RV induces biochemical changes in the infected cells as evidenced by the increased production of cytokines that could play a role in the recruitment and activation of inflammatory cells into the airway of infected individuals. Of these cytokines, IL-1 is an autocrine mechanism of enhanced ICAM-1 expression in RV-infected submucosal gland cells. This model system provides a valuable tool for RV infection of submucosal gland cells and should yield useful insights into the initial stages of inflammation and host immune responses in the microenvironment of the respiratory mucosa associated with the pathogenesis of RV infections.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Walter E. Finkbeiner (University of California, Davis) for providing the A1F8 and B1D8 antibodies; to Akira Ohmi, Michiko Okamoto, and Minako Tada for technical assistance; and to Grant Crittenden for reading the manuscript.
![]() |
FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Sasaki, Dept. of Geriatric Medicine, Tohoku University School of Medicine, Aoba-ku Seiryo-machi 1-1, Sendai 980-8574, Japan.
Received 8 June 1998; accepted in final form 10 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akira, S.,
T. Hirano,
T. Taga,
and
T. Kishimoto.
Biology of multifunctional cytokines: IL-6 and related molecules (IL-1 and TNF).
FASEB J.
4:
2860-2867,
1990[Abstract].
2.
Baggiolini, M.,
A. Walz,
and
S. L. Kunkel.
Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.
J. Clin. Invest.
84:
1045-1049,
1989[Medline].
3.
Bardin, P. G.,
S. L. Johnston,
G. Sanderson,
B. S. Robinson,
M. A. Pickett,
D. J. Fraenkel,
and
S. T. Holgate.
Detection of rhinovirus infection of the nasal mucosa by oligonucleotide in situ hybridization.
Am. J. Respir. Cell Mol. Biol.
10:
207-213,
1994[Abstract].
4.
Becker, S.,
H. S. Koren,
and
D. C. Henke.
Interleukin-8 expression in normal nasal epithelium and its modulation by infection with respiratory syncytial virus and cytokines tumor necrosis factor, interleukin-1, and interleukin-6.
Am. J. Respir. Cell Mol. Biol.
8:
20-27,
1993[Medline].
5.
Beckstead, J. H.
Optimal antigen localization in human tissues using aldehyde-fixed plastic-embedded sections.
J. Histochem. Cytochem.
33:
954-958,
1985[Abstract].
6.
Borum, P.,
L. Olsen,
B. Winther,
and
N. Mygind.
Ipratropium nasal spray: a new treatment for rhinorrhea in the common cold.
Am. Rev. Respir. Dis.
123:
418-420,
1981[Medline].
7.
Busse, W. W.,
C. A. Swenson,
E. C. Borden,
M. W. Treuhaft,
and
E. C. Dick.
Effect of influenza A virus on leukocyte histamine release.
J. Allergy Clin. Immunol.
71:
382-388,
1983[Medline].
8.
Carson, J. L.,
A. M. Collier,
and
S. S. Hu.
Acquired ciliary defects in nasal epithelium of children with acute viral upper respiratory infections.
N. Engl. J. Med.
312:
463-468,
1985[Abstract].
9.
Cate, T. R.,
R. B. Couch,
W. F. Fleet,
W. R. Griffith,
P. J. Gerone,
and
V. Knight.
Production of tracheobronchitis in volunteers with rhinovirus in small particle aerosol.
Am. J. Epidemiol.
81:
95-105,
1965.
10.
Couch, R. B.
Rhinoviruses.
In: Virology, edited by B. N. Fields,
D. M. Knipe,
R. M. Chanock,
M. S. Hirsch,
J. L. Melnick,
T. P. Monath,
and B. Roizman. New York: Raven, 1990, p. 607-629.
11.
Fraenkel, D. J.,
P. G. Badin,
G. Sanderson,
F. Lampe,
S. L. Johnston,
and
S. T. Holgate.
Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects.
Am. J. Respir. Crit. Care Med.
151:
879-886,
1995[Abstract].
12.
Gern, J. E.,
R. Vrtis,
E. A. B. Kelly,
E. C. Dick,
and
W. W. Busse.
Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism.
J. Immunol.
157:
1605-1612,
1996[Abstract].
13.
Greve, J. M.,
G. Davis,
A. M. Meyer,
C. P. Forte,
S. C. Yost,
C. W. Marlor,
M. E. Kamarck,
and
A. McClelland.
The major human rhinovirus receptor is ICAM-1.
Cell
56:
839-847,
1989[Medline].
14.
Gwaltney, J. M., Jr.,
J. Park,
R. A. Paul,
D. A. Edelman,
R. R. O'Connor,
and
R. B. Turner.
Randomized controlled trial of clemastine fumarate for treatment of experimental rhinovirus colds.
Clin. Infect. Dis.
22:
656-662,
1996[Medline].
15.
Hamory, B. H.,
J. O. Hendley,
and
J. M. Gwaltney, Jr.
Rhinovirus growth in nasal poly organ culture.
Proc. Soc. Exp. Biol. Med.
155:
577-582,
1977.
16.
Horn, M. E. C.,
S. E. Reed,
and
P. Taylor.
Role of viruses and bacteria in acute wheezy bronchitis in childhood: a study of sputum.
Arch. Dis. Child.
54:
587-592,
1979[Abstract].
17.
Hughes, J. H.,
M. Mitchell,
and
V. V. Hamparian.
Rhinoviruses: kinetics of ultraviolet inactivation and effects of UV and heat on immunogenicity.
Arch. Virol.
61:
313-319,
1979[Medline].
18.
Johnston, S. L.,
P. K. Pattemore,
G. Sanderson,
S. Smith,
F. Lampe,
L. Josephs,
P. Symington,
S. O'Toole,
S. H. Myint,
D. A. J. Tyrrell,
and
S. T. Holgate.
Community study of role of viral infections in exacerbations of asthma in 9-11 year old children.
Br. Med. J.
310:
1225-1229,
1995
19.
Johnston, S. L.,
G. Sanderson,
P. K. Pattemore,
S. Smith,
P. G. Bardin,
C. B. Bruce,
P. R. Lambden,
D. A. J. Tyrrell,
and
S. T. Holgate.
Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms.
J. Clin. Microbiol.
31:
111-117,
1993[Abstract].
20.
Larsen, C. G.,
A. O. Anderson,
E. Appella,
J. J. Oppenheim,
and
K. Matsushima.
The neutrophil-activating protein (NAP-1) is also chemotactic for T lymphocytes.
Science
243:
1464-1466,
1989[Medline].
21.
Levandowski, R. A.,
C. W. Weaver,
and
G. G. Jackson.
Nasal-secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection.
J. Med. Virol.
25:
423-432,
1988[Medline].
22.
Lopez, A. F.,
D. J. Williamson,
J. R. Gamble,
C. G. Begley,
J. M. Harlan,
S. J. Klebanoff,
A. Waltersdorph,
G. Wong,
S. C. Clark,
and
M. A. Vadas.
Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival.
J. Clin. Invest.
78:
1220-1228,
1986[Medline].
23.
Nicholson, K. G.,
J. Kent,
and
D. C. Ireland.
Respiratory viruses and exacerbations of asthma in adults.
Br. Med. J.
307:
982-986,
1993[Medline].
24.
Noah, T. L.,
and
S. Becker.
Respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L472-L478,
1993
25.
Numazaki, Y.,
T. Oshima,
A. Ohmi,
A. Tanaka,
Y. Oizumi,
S. Komatsu,
T. Takagi,
M. Karahashi,
and
N. Ishida.
A microplate method for isolation of viruses from infants and children with acute respiratory infections.
Microbiol. Immunol.
31:
1085-1095,
1987[Medline].
26.
Ohrui, T.,
M. Yamaya,
K. Sekizawa,
N. Yamada,
T. Suzuki,
M. Terajima,
S. Okinaga,
and
H. Sasaki.
Effects of rhinovirus infection on hydrogen peroxide-induced alterations of barrier function in the cultured human tracheal epithelium.
Am. J. Respir. Crit. Care Med.
158:
241-248,
1998
27.
Patel, J. A.,
M. Kunimoto,
T. C. Sim,
R. Garofalo,
T. Eliott,
S. Baron,
O. Ruuskanen,
T. Chonmaitree,
P. L. Ogra,
and
F. Schmalstieg.
Interleukin-1 mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus.
Am. J. Respir. Cell Mol. Biol.
13:
602-609,
1995[Abstract].
28.
Reid, L.
Measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis.
Thorax
15:
132-141,
1960.
29.
Sambrook, J.,
E. Fritsch,
and
T. Maniatis.
Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
30.
Sommerhoff, C. P.,
and
W. E. Finkbeiner.
Human tracheobronchial submucosal gland cells in culture.
Am. J. Respir. Cell Mol. Biol.
2:
41-50,
1990[Medline].
31.
Subauste, M. C.,
D. B. Jacoby,
S. M. Richards,
and
D. Proud.
Infection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure.
J. Clin. Invest.
96:
549-557,
1995[Medline].
32.
Terajima, M.,
M. Yamaya,
K. Sekizawa,
S. Okinaga,
T. Suzuki,
N. Yamada,
K. Nakayama,
T. Ohrui,
T. Oshima,
Y. Numazaki,
and
H. Sasaki.
Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L749-L759,
1997
33.
Tomassini, J. E.,
D. Graham,
C. M. DeWitt,
D. W. Lineberger,
J. A. Rodkey,
and
R. J. Colonno.
cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1.
Proc. Natl. Acad. Sci. USA
86:
4907-4911,
1989[Abstract].
34.
Turner, R. B.,
J. O. Hendley,
and
J. M. Gwaltney, Jr.
Shedding of infected ciliated epithelial cells in rhinovirus colds.
J. Infect. Dis.
145:
849-853,
1982[Medline].
35.
Winther, B.,
J. M. Gwaltney,
and
J. O. Hendley.
Respiratory virus infection of monolayer cultures of human nasal epithelial cells.
Am. Rev. Respir. Dis.
141:
839-845,
1990[Medline].
36.
Yamaya, M.,
W. E. Finkbeiner,
and
J. H. Widdicombe.
Ion transport by cultures of human tracheobronchial submucosal glands.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L485-L490,
1991
37.
Zhu, Z.,
W. Tang,
A. Ray,
Y. Wu,
O. Einarsson,
M. L. Landry,
J. Gwaltney, Jr.,
and
J. A. Elias.
Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappaB-dependent transcriptional activation.
J. Clin. Invest.
97:
421-430,
1996