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INTRODUCTION |
Human rhinoviruses
(HRV)1 are the most frequent
cause of upper respiratory tract infections known as the "common
cold." Although these infections are generally mild and
self-limiting, they inflict a heavy economical burden due to high loss
of productivity and medical costs (1). Currently, there is no effective
treatment for HRV infections; over the counter cold remedies only
alleviate the symptoms but do not eradicate the virus.
Primarily, HRV target epithelial cells for attachment and entry. These
cells express intercellular adhesion molecule 1 (ICAM-1), the receptor
for 90% of HRV serotypes (2). Both this major group of HRV and the
10% of HRV that use alternative receptors for cell attachment enhance
cell surface ICAM-1 expression (3). This glycoprotein, belonging to the
immunoglobulin supergene family, consists of five Ig-like domains (4);
domains 1 and 2 have been shown to fit snugly in a key-lock
relationship into reciprocal canyons on the HRV shell (5). In addition,
to this critical role as a docking molecule during HRV infection,
ICAM-1 through separate domains with its cognate ligand LFA-1
(CD18/CD11a) drives the migration of immune-effector cells to sites of
inflammation (6). While most studies refer to the membranous form of
ICAM-1, a soluble form (sICAM-1) has also been described (7). The
molecular mass of sICAM-1 is similar to the molecular mass of
the extracellular domain of ICAM-1 (80-114 kDa) depending on the level
of glycosylation, suggesting that this soluble circulating form of
ICAM-1 consists of most of the extracellular domain of membranous
ICAM-1 (7). Several circulating isoforms of sICAM-1 have also been
detected of 240, 430, and >500 kDa in size, indicating that sICAM-1
may circulate in a complexed form either with itself or with other proteins (7-8). While the exact origin of sICAM-1 is unclear, sICAM-1
may be produced directly from the membrane-bound form by proteolytic
cleavage (9-10) or produced independently by an alternative splicing
mechanism (11). What role is played by soluble ICAM-1 in disease
pathogenesis remains to be elucidated.
Previous studies have demonstrated that pro-inflammatory cytokines can
alter the expression of mICAM-1 (3, 12-14) and sICAM-1 (15-16). In
addition, HRV infection has been shown to significantly up-regulate the
expression of its membrane-bound receptor ICAM-1 on the surface of
epithelial cells (3, 12-14) leading to an increase in epithelial cell
infectivity (12). However, there is evidence indicating that sICAM-1
may have the opposite effect because it possesses antiviral properties
both in vitro (17) and in vivo (18-19).
Therefore, the dynamic inter-relationship between mICAM-1 and sICAM-1
forms may have a critical bearing on the pathogenesis as well as course
of HRV infection. Thus, there is a need for a better understanding of
the interaction between mICAM-1 and sICAM-1, leading to potential
targets for therapeutic modulation of the course of HRV infection.
To investigate this hypothesis, a series of studies were undertaken to
establish the presence of distinct mRNA transcripts coding for
mICAM-1 and sICAM-1 in an in vitro airway epithelial cell
model and to determine whether HRV has the ability to modulate the two
forms of ICAM-1 and how this affects epithelial cell infectivity. Having found that epithelial cells express the two ICAM-1 forms and
that HRV could selectively modulate membrane and soluble ICAM-1 expression in an inverse fashion to promote/propagate infection, we
explored the potential intracellular mechanisms influencing this
differential modulation to identify potential targets for antiviral therapy.
ICAM-1 is a rapid response gene with a complex pattern of
regulation (20). Numerous second messenger signaling pathways involved
in the activation of the ICAM-1 gene have been
identified (20-21). A recent study demonstrated that HRV up-regulates
membrane-bound ICAM-1 expression in airway epithelial cells via an
NF
B-dependent mechanism (3). We investigated this
intracellular signaling pathway further and examined the effect of HRV
on tyrosine kinase phosphorylation in airway epithelial cells. In
addition, we utilized inhibitors of gene transcription and protein
synthesis to investigate potential mechanisms responsible for the
modulation of sICAM-1 production/release during HRV infection.
Our results demonstrate that HRV selectively induces mICAM-1
expression on epithelial cells, at least in part, through a tyrosine kinase-dependent pathway, while, HRV influence on sICAM-1
release may involve the down-regulation/inhibition of proteolytic
enzymes associated with the cleavage of mICAM-1 from the epithelial
cell surface. The interaction of HRV with these intracellular molecular pathways controlling mICAM-1/sICAM-1 ratios will need to be further dissected, specifically for the development of new anti-HRV strategies aimed at either halting progression or reversing host cell infectivity.
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MATERIALS AND METHODS |
Epithelial Cell Culture--
Normal human bronchial epithelial
cells (NHBE) were obtained from Clonetics Corp., Walkersville, MD. The
donor was a middle-aged male Caucasian. NHBE cells were cultured in
small airway basal medium (SABM) supplemented with epidermal growth
factor (25 ng/ml), hydrocortisone (0.5 µg/ml), insulin (5 µg/ml),
transferrin (10 µg/ml), epinephrine (0.5 µg/ml), triiodothyronine
(6.5 ng/ml), bovine pituitary extract (52 µg/ml), retinoic acid (0.1 ng/ml), gentamicin (50 mg/ml), amphotericin B (50 µg/ml) at 37 °C
in humidified air containing 5% CO2. All reagents were
obtained from Clonetics Corp. In subsequent experiments, NHBE cells
were seeded in 6-well plates at a density of 100,000 cells/well and
utilized when 70-80% confluent.
Viral Stocks--
The main rhinovirus seed (HRV-14) was kindly
donated by J. Kent (University of Leicester). A stock solution of
HRV-14 was generated by infecting confluent monolayers of HeLa Ohio
cell line as described previously (Sethi et al., Ref. 12).
Briefly, confluent monolayers of Hela cells were inoculated with a
known dilution (102.5, TCID50/ml) of
HRV-14 and incubated for 90 min at 34 °C in humidified air
containing 5% CO2, after which, cells were cultured until the cytopathic effect (CPE) was >80%. Medium containing virus was
centrifuged at 600 × g for 10 min, after which the
viral suspension was stored at
80 °C until required.
Viral Purification--
Prior to use viral stocks were purified
using a sucrose gradient. 20 µg/ml RNase A (Sigma) was added to the
viral suspension and incubated at 35 °C for 20 min. 1% sodium
sarkosyl (Sigma) and 2-mercaptoethanol (1 µg/ml) were added to the
RNase-treated viral suspension. This was then overlaid on 1 ml of
purification solution (20 mM Tris acetate, 1 M
NaCl, 30% w/v sucrose) and centrifuged at 200,000 × g
for 5 h at 16 °C. The supernatant was discarded, and the
resulting virus pellet was resuspended in medium and stored at
80 °C until required.
HRV-14 Infection of Epithelial Cells--
Once cell monolayers
were 70-80% confluent the culture medium was removed, and the cells
were inoculated with HRV-14 102.5 TCID50/ml for
90 min at 34 °C, 5% CO2/air. The cells were washed, and
maintenance medium was added to sustain cell growth. The gene and
protein expression of the two ICAM-1 forms were measured simultaneously at 0, 8, 24, and 96 h postinfection. 0 h represents the point immediately after the 90-min inoculation period; subsequent time points
(8-96 h) are taken from this 0 h.
Measurement of mICAM-1 Protein Expression--
Membrane-bound
ICAM-1 expression was evaluated at 0, 8, 24, and 96 h post-HRV-14
infection. At each time point, cells from 6-well plates were collected
via trypsinization and centrifugation; 6 cytospins for each
experimental condition at each time point were prepared for
immunostaining. The remaining cells were utilized for RNA extraction,
cDNA synthesis, and reverse transcription (RT)-PCR. Internal
controls consisting of unstimulated and uninfected cells were set up at
each time point to allow comparisons between controls and treated
cells. A cytokeratin immunoglobulin IgG-specific monoclonal antibody
(Sigma) was used to confirm the epithelial origin of NHBE cell lines.
Surface ICAM-1 was semiquantified using a 3-step indirect
immunoenzymatic labeling method (22) and modified as described
previously (12-13). Briefly, exogenous peroxidase staining was blocked
using 2% bovine serum albumin/phosphate-buffered saline solution. NHBE
cells were incubated with ICAM-1 monoclonal antibody at a concentration
of 5 µg/ml, (R1/1.1, IgG, Boehringer Ingelheim) at room temperature
for 30 min. Cells were then washed using a washing buffer (Tris stock
solution, 0.05 mol/liter, pH 7.4, NaCl, 0.9% saline solution), and
incubated with rabbit anti-mouse IgG conjuagated to peroxidase at a
concentration of 1 mg/ml (Dako) for 30 min; the cells were then washed
again. A third antibody, swine anti-rabbit IgG also conjugated to
peroxidase, (DAKO), was then added at a concentration of 0.8 mg/ml to
amplify the staining intensity. The cells were then incubated with the
substrate 3,3-diaminobenzidine tetrahydrochroride (0.6 mg/ml, Sigma)
and stained with Mayers Hemalum solution (BDH). The cells were
incubated with an anti-mouse IgG antibody (Coulter clone) at a
concentration of 10 µg/ml, which acted as a negative control.
To avoid observer bias, the cytospins were scored by two independent
observers (A. Bianco and S. Whiteman); a mean of three readings of each
slide was performed. Two cytospins for each experimental condition per
time point were assessed at ×400 magnification with a light microscope
(Olympus CH-2 microscope, Olympus Optical Co., Ltd., Tokyo). 300 cells
per microscopic field were counted and surface ICAM-1 on epithelial
cells was assessed using a 5 point scoring scale based on the intensity
of staining and appearance of the nucleus: 0, gray/brown; 1, light
brown; 2, medium brown; 3, medium/dark brown; 4, dark brown; in grades
0-2 the nucleus appears well defined, in 3-4 the nucleus is partially
or fully obliterated. The number of cells scored in each grade was then multiplied with the respective grade index, and the resulting values
summed. The final result was expressed as the Pox score (12-13, 22),
defined as the difference between the sum of the specific
and background staining: [(a × 0) + (b × 1) + (c × 2) + (d × 3) + (e × 4)]
value for the control
slide = POX score, where each letter represents the number of
cells scored in the respective grade. The coefficient of variability of
the differences between the counts obtained from all slides by both
observers was between 4 and 12%; and that between the two observers
for each time point was less than 5%.
Measurement of sICAM-1 Protein in Cell Culture
Supernatants--
Soluble ICAM-1 expression was also evaluated at 0, 8, 24, and 96 h post-HRV-14 infection. Internal controls
consisting of unstimulated and uninfected cells were setup at each time
point to allow comparisons between controls and treated cells. Cell culture supernatants retrieved from each experimental condition at each
time point were assayed for soluble ICAM-1 using a commercially available ELISA kit (BioSource International, CA). The minimum detectable level of human soluble ICAM-1 (hsICAM-1) was <0.04 ng/ml.
100 µl of undiluted cell culture supernatant or standard were
utilized in the assay, which was performed in accordance with the
manufacturer's guidelines.
RNA Extraction and cDNA Synthesis--
Total RNA was
extracted from NHBE cells at 0, 8, 24, and 96-h postinfection using
Trizol (Invitrogen) according to the manufacturer's guidelines, and
cDNA was synthesized from 2 µg of RNA. cDNA synthesis was
conducted in a reaction mixture containing 20 pmol oligo(dT) primer,
5× buffer (50 mM, pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.5 mMdNTP mixture, 0.5 units of
RNase inhibitors, and 200 units of MMLV reverse transcriptase; the
total reaction volume was 20 µl. All cDNA synthesis reagents were
obtained from Clontech. This was then incubated at
42 °C for 1 h after which the reverse transcriptase and DNase
were heat-inactivated at 94 °C for 5 min. The cDNA was then
diluted to a final volume of 100 µl and stored at
80 °C for
RT-PCR.
Detection of Membrane and Soluble ICAM-1 Gene Expression using
RT-PCR--
Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used
as a control for cDNA synthesis and RT-PCR. Primers used to detect G3PDH were 5'-TGA AGG TCG GAG TCA GA-3' (sense) and CAT GTG GGC CAT GAG
GTC CAC CAC (antisense). Primers for the detection of mICAM-1 and
sICAM-1 were based on those described previously by Wakatsuki et
al. (11). The sequence of the forward primer used to detect
mICAM-1 was 5'-CAA GGG GAG GTC ACC CGC GAG GTG-3' and 5'-CAA GGG AGG
TCA CCC GCG AGC C-3'. Both primers were used in combination with a
common reverse primer with the following sequence 5'-TGC AGT GCC CAT
TAT GAC TG-3'. These primer pairs encompass the transmembrane domain of
ICAM-1. The primers used to detect HRV were CGG ACA CCC AAA GTA G
(sense) and GCA CTT CTG TTT CCC C (antisense). The RT-PCR consisted of
25 pmol of primers, 200 µM dNTPs, 1.5 mM
MgCl2, 5 µl 10× PCR buffer, and 2.5 units of Amplitaq
Gold (PerkinElmer Life Sciences) in a 50-µl reaction mixture in a
thermal cycler (PTC 200 Pielter Thermal cycler) under the following
conditions: 95 °C for 12 min, 94 °C for 1 min and 15 s
(denaturation step), 60 °C (G3PDH) or 65 °C (ICAM-1) for 1 min
and 15 s (annealing step) and 72 °C for 1 min (extension step)
for a total of 30 cycles (G3PDH) or 35 cycles (ICAM-1), after which a
final extension step was performed at 72 °C for 10 min. RT-PCR
products were resolved using 3% metaphor agarose (Flowgen) gel in TBE
buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, Sigma). Gels were visualized using ethidium
bromide and UV light and analyzed densitometrically (Model GS-670,
BioRad) using Molecular Analyst (version 1.5). Restriction
endonucleases were used to confirm the size of the RT-PCR products.
Membrane-bound ICAM-1 RT-PCR products were digested to give product
sizes of 45 and 57 base pairs, and soluble ICAM-1 RT-PCR products were digested at the site of the deletion to give 63 and 20 bp products. In
addition, to confirm the presence of the 19-bp deletion, RT-PCR amplicons were sequenced using an ABI PRISM automated sequencer model 310.
Viral Titer Assay--
The TCID50 method was used to
calculate the concentration of the virus in cell culture supernatants
at 0, 8, 24, and 96 h postinfection. Serial dilutions of cell
culture supernatants were incubated in cell monolayers in 96-well
plates for 5 days at 34 °C in humidified air containing
5%CO2. The presence of cytopathic effect (CPE) in the
wells was used to calculate the TCID50 using the Karber
formula (12-14). Furthermore, cell infectivity was confirmed by
detecting the presence of HRV RNA within the cell using RT-PCR (23).
Prevention of Virus-Receptor Binding--
To confirm the changes
observed in ICAM-1 expression were true effects of HRV and not due to
soluble factors within the viral inoculum, separate studies were
designed using anti-ICAM-1 monoclonal antibodies to block HRV
attachment and subsequent infection. NHBE cell monolayers were washed
and incubated with anti-ICAM-1 monoclonal antibodies (mAb) (R1/1.1
Boerhinger Ingelheim) at separate concentrations of 4, 8, and 16 µg/ml for 1 h at 37 °C under 5% CO2-humidified air (24). After which the anti-ICAM-1 mAb solution was removed, and
cell monolayers were washed and inoculated with HRV-14 at a
concentration of 102.5 TCID50/ml for 90 min at
34 °C under 5% CO2-humidified air. After 90 min the
viral inoculum was removed, cell monolayer washed, and maintenance
medium was replaced. Gene expression of both ICAM-1 forms was evaluated
using RT-PCR at 24 h, because this time point was previously shown
to reflect an optimum response in gene expression. Corresponding viral
titers were also measured.
Inhibition of de Novo Protein Synthesis--
To assess the level
at which HRV-14 regulates the expression of both ICAM-1 receptors,
separate cell cultures were preincubated with cycloheximide, an
inhibitor of de novo protein synthesis at a concentration of
10 µg/ml for 2 h at 37 °C in humidified air containing 5%
CO2. Cycloheximide is widely used as an inhibitor of
protein synthesis and has an effect at 10 µg/ml (25). The cell
monolayers were then washed and infected with HRV-14 as described above. Membrane and soluble ICAM-1 protein levels were assessed as
described above.
Inhibition of Gene Transcription--
Actinomycin D, an
inhibitor of gene transcription was used to assess the effect of HRV-14
on ICAM-1 gene transcription. NHBE cells were incubated with
actinomycin D at a concentration of 10 µg/ml for 2 h at 37 °C
in humidified air containing 5% CO2. 10 µg/ml was
identified as the optimum dose in previous dose response experiments
(data not shown). The cell monolayers were then washed and infected
with HRV-14 as described above. Membrane-bound and soluble ICAM-1 were
assessed using RT-PCR and semiquantified using densitometry (Model
GS-670, BioRad) and Molecular Analyst Software (version 1.5).
Investigation of the Role of Tyrosine Kinases in HRV Induction of
ICAM-1 Expression--
The involvement of tyrosine kinase in the
HRV-driven up-regulation of mICAM-1 gene expression was investigated
using Western blot. NHBE cells were cultured in SABM (detailed under
"Materials and Methods") and infected with HRV-14
(TCID50 102.5) for the indicated times (0-30
min, 0 min represents the viral inoculum placed on the cell monolayer
and then immediately removed). Cell were also pretreated with genistein
(50 and 100 µM), an inhibitor of tyrosine kinase for
1 h at 37 °C in 5% CO2-humidified air. NHBE cells
were lysed using a lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 137 mM sodium chloride,
10% glycerol, 1 mM sodium orthovanadate, 2 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 0.15 units/ml aprotinin. All reagents were molecular biology grade and obtained from Sigma. The cells were
placed on ice for 20 min, and total protein was collected by
centrifugation. Total protein was assayed using a commercially available kit based on the Lowry assay (Bio-Rad). 25 µg of reduced protein samples were electrophoresed on 12.5% SDS-PAGE and transferred to nitrocellulose membranes (sandwiches, 0.45-µm pore size Novex, San
Diego). Molecular weight markers and epidermal growth factor receptor
(control) were run with the samples. Membranes were blocked with 10%
(w/v) low fat milk for 1 h in TBS-T and probed for 2 h with
mouse anti-human phosphotyrosine kinase (clone 4G10, Upstate Technology) diluted 1:3000 in TBS-T. Membranes were incubated with a
horseradish peroxidase-conjugated rabbit anti-mouse antibody (Dako)
diluted 1:40,000 in TBS-T for 1 h. The ECL system was used for
detection (Amersham Biosciences). The membranes were reprobed with
a control mouse IgG as a negative control. The membranes were analyzed
densitometrically (Model GS-670, BioRad) and Molecular Analyst Software
(version 1.5).
Statistical Analysis--
Each experiment was performed three
times. Data were expressed as means ± S.E., and comparisons
between experimental conditions and controls were performed by paired
Student's t test. Probability values < 0.05 were
considered significant.
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RESULTS |
Epithelial Cell Expression of Two Distinct Forms of mRNA Coding
for ICAM-1--
Two distinct mRNA transcripts were observed in
NHBE cells after RT-PCR (Fig. 1). These
RT-PCR products corresponded with those observed by Wakatsuki et
al. (11) with RT-PCR products of 102 and 83 bp for mICAM-1 and
sICAM-1, respectively. Product sizes were confirmed by restriction
enzyme digestion (data not shown).

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Fig. 1.
Representative gel illustrating mICAM-1 and
sICAM-1 mRNA transcripts at 24-h post-HRV inoculation in control
uninfected and HRV-14-infected NHBE cells demonstrated using
RT-PCR. The housekeeping gene G3PDH is also shown.
Semiquantification using densitometry was carried out; data are
expressed as the ratio of ICAM-1 normalized to G3DPH (± S.E.).
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Influence of HRV-14 on mICAM-1 Expression--
mICAM-1 surface
protein was constitutively expressed in NHBE cells (Fig.
2A). HRV-14 infection induced
an up-regulation in both surface mICAM-1 protein and gene expression
(Fig. 2, A and B). Surface mICAM-1 protein
expression on NHBE cells increased by 2.5-fold over baseline at 8-h
post-HRV-14 infection (*, p < 0.001, Fig.
2A). The enhanced mICAM-1 protein levels were sustained throughout the 96-h period of infection and was accompanied by a 2-fold
increase in mICAM-1 gene expression (normalized to the housekeeping
gene G3PDH) only at 24 h through to 96 h (*,
p = 0.02, Fig. 2B).

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Fig. 2.
Effect of HRV-14 (TCID50
102.5 for 90 min) on mICAM-1 protein expression
(A), mICAM-1 gene expression (B) in
NHBE cells over the study period (0-96 h; time 0 h represents the
point immediately after the 90 min viral inoculation period).
mICAM-1 protein expression was measured using immunocytochemistry and
expressed as a Pox score. Data are mean ± S.E. of three separate
experiments (*, p < 0.001 compared with control
uninfected cells). Gene expression of ICAM-1 receptors was
semiquantified using RT-PCR and densitometry and expressed as a ratio
to the housekeeping gene G3PDH. Data are mean ± S.E.
of three separate experiments (B, *, p < 0.02 compared with controls). Control uninfected cells are
represented by filled circles and HRV-infected cells are
represented by open squares.
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Influence of HRV-14 on sICAM-1 Expression--
Basal
sICAM-1 protein release in cell culture supernatants increased at
8 h and thereafter decreased in a time-dependent
manner. However, no sICAM-1 protein was detected in cell culture
supernatants retrieved from HRV-infected cell lines at any time point
studied. Gene analysis showed that HRV-14 infection induced a
persistent down-regulation in sICAM-1 gene expression in NHBE cells
over the study period (0-96 h); with sICAM-1 mRNA levels
decreasing to half-basal mRNA levels at 24 h (*,
p < 0.001, Table I).
Fig. 1 illustrates the simultaneous changes in membrane and soluble ICAM-1 gene expression during HRV infection of NHBE cells.
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Table I
Effect of HRV-14 on sICAM-1 gene expression
sICAM-1 gene expression was semi quantified using RT-PCR and
densitometry and expressed as a ratio to the housekeeping gene
G3PDH. Data are mean ± S.E. of three separate experiments
(*, p < 0.05 compared to control).
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Analysis of Recovered Viral Titers--
A steep increase in viral
titers was observed at 8 h, where viral titers reached 6-fold (*,
p < 0.01, Fig.
3A). Viral titers remained
elevated through to 96 h. The presence of HRV RNA within the NHBE
cells was confirmed by RT-PCR (Fig. 3B). RT-PCR produced an
amplicon of 383-392 bp, which was further digested using
BglI to give a product of 190 base pairs confirming
the presence of rhinoviral RNA (data not shown).

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Fig. 3.
Measurement of epithelial cell infectivity by
measuring viral titers (A) and HRV-14 RNA
(B). Viral titers in recovered cell culture
supernatants were measured using the TCID50 and are
expressed a log TCID50/ml. Data are mean ± S.E. of
three separate experiments (*, p < 0.01 compared with
viral titers at 0 h). B is a representative gel
illustrating HRV-14 RNA within NHBE cells at 24 and 96 h.
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Prevention of Virus-Receptor Binding--
In order to confirm that
the observed changes in epithelial cell membrane and soluble ICAM-1
expression, reflected the direct influence of HRV-14, separate blocking
experiments were carried out using anti-ICAM-1 mAb. NHBE cultures were
incubated with ICAM-1 mAb prior to HRV-14 infection.
mICAM-1 and sICAM-1 gene expression were evaluated 24-h
post-HRV-14 infection. Viral titers in retrieved cell culture
supernatants were also measured. Incubation of control uninfected
cultures with ICAM-1 mAb had no significant effect on the gene
expression of mICAM-1 and sICAM-1 compared with equivalent untreated
cultures (data not shown). Preincubation with ICAM-1 mAb at 4 µg/ml
resulted in a decrease in HRV-14-induced gene expression of mICAM-1 (*,
p < 0.02 Fig.
4A) but had no significant
effect on the gene expression of sICAM-1. Preincubation with 8 µg/ml and 16 µg/ml ICAM-1 mAb also resulted in a decrease in mICAM-1 expression. In addition, these higher concentrations also produced small but significant increases in sICAM-1 expression (*,
p < 0.02, Fig. 4A), suggesting the effects
of HRV were inhibited. ICAM-1 mAb decreased viral titers in cell
culture supernatants in a dose-dependent manner (Fig.
4B, *, p < 0.02).

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Fig. 4.
Effect of ICAM-1 mAb (4-16
µg/ml) on mICAM-1 (black bars) and
sICAM-1 (gray bars) gene expression
(A) and viral titers (B). ICAM-1
gene expression was semiquantified at 24 h using RT-PCR and
densitometry and expressed as a ratio to the housekeeping gene
G3PDH. Data are mean ± S.E. of three separate
experiments (*, p < 0.05 compared with mICAM-1
expression of untreated infected cells; **, p < 0.02 compared with sICAM-1 expression of untreated infected cells). Viral
titers in recovered cell culture supernatants were measured using the
TCID50 and are expressed a log TCID50/ml. Data
are mean ± S.E. of three separate experiments (*,
p < 0.02 compared with viral titers from infected
untreated cells).
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Inhibition of de Novo Protein Synthesis--
Treatment of NHBE
cells with cycloheximide (10 µg/ml) resulted in an inhibition of
HRV-induced mICAM-1 protein expression at 0 and 8 h, suggesting
that HRV-14 induces de novo protein synthesis of mICAM-1.
However cycloheximide had no significant effect on mICAM-1 protein
expression at 24 and 96 h (data not shown). This may be caused by
the depletion of cycloheximide in the culture medium as it has a short
half-life. In contrast, sICAM-1 release from the same HRV-infected NHBE
cells was not affected by cycloheximide, implying that the regulation
of sICAM-1 release from infected cells is not dependent on de
novo protein synthesis.
Inhibition of Gene Transcription--
Treatment of NHBE cells with
actinomycin D (10 µg/ml) resulted in a complete inhibition of the
expected HRV-induced mICAM-1 protein expression at 8, 24, and 96 h
(*, p < 0.001, Fig.
5A). In contrast, actinomycin
D enhanced sICAM-1 protein release from HRV-infected cells at all
experimental time points post-HRV inoculation. These observations
indicate that HRV may down-regulate sICAM-1 release by inhibiting gene
transcription of a suppressor/inhibitor of an enzyme responsible for
the cleavage of mICAM-1 (*, p < 0.001, Fig.
5B).

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Fig. 5.
Effect of actinomycin D (10 µg/ml) on mICAM-1 (A) and sICAM-1
protein expression (B) in HRV-14-infected cells at
0-96 h. mICAM-1 protein expression was measured using
immunocytochemistry and expressed as a Pox score. Data are mean ± S.E. of three separate experiments (*, p < 0.001 compared with control uninfected cells). Control uninfected cells are
represented by black bars. Control uninfected cells
preincubated with actinomycin D (abbreviated as Ac D) are
represented by black striped bars, HRV-infected cells are
represented by gray bars, and HRV-infected cells
preincubated with AcD are represented by gray checked bars.
sICAM-1 protein release in cell culture supernatants were measured
using ELISA. Data are mean ± S.E. of three separate experiments
(*, p < 0.001 compared with untreated HRV-14 infected
cells). HRV-infected cells are represented by gray bars and
HRV-infected cells preincubated with AcD are represented by black
bars.
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Investigation of the Role of Tyrosine Kinases in HRV Induction of
ICAM--
1 Expression
Previous studies have demonstrated that the
malarial parasite Plasmodium falciparum utilizes and
up-regulates ICAM-1 expression via a tyrosine
kinase-dependent mechanism (26). We therefore sought to
determine whether HRV-14 also acts on ICAM-1 expression via this
mechanism. Phosphorylation of tyrosine kinase was assessed using
Western analysis. HRV-14 infection of NHBE cells induced the de
novo phosphorylation of a number of cellular substrates (Fig.
6A). Densitometry of three
separate blots was performed, and the data are expressed as a
percentage increase in tyrosine kinase phosphorylation in HRV-infected
cells compared with control uninfected cells at each time point. The
predominant substrates included a band at 85 kDa (Fig. 6B)
and at 200 kDa (Fig. 6C). HRV-14 induced a rapid increase in
tyrosine kinase phosphorylation occurring within 5 min of infection,
where tyrosine kinase phosphorylation of the 85- and 200-kDa substrates
increased by 80 and 120%, respectively, compared with uninfected to
control uninfected cells (*, p < 0.01). This
activation of the 200-kDa substrate was reduced by 30-min postviral
inoculation, while the 85-kDa protein remained activated up to 30 min
(*, p = 0.05). The observed HRV-induction of tyrosine
kinase phosphorylation was completely inhibited by preconditioning
cells with 50 and 100 µM genistein (data not shown). In
addition, incubation of these NHBE cells with genistein down-regulated
the HRV-induced increase in mICAM-1 protein on the cell surface (*,
p < 0.001, Fig. 7).

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Fig. 6.
Western blot of phosphorylated tyrosine
kinase activity in control uninfected NHBE cells and HRV-infected cells
over a time course. M represents the molecular weight
marker; control cells at 0 min (lane 1), infected cells at 0 min (lane 2), infected cells at 5 min (lane 3),
control cells at 5 min (lane 4), control cells at 15 min
(lane 5), infected cells at 15 min (lane 6),
infected cells at 30 min (lane 7), and control cells at 30 min (lane 8). Time 0 min represents the viral inoculum
placed onto the cell monolayer and then immediately removed; subsequent
time points are taken from this point. This blot is typical of three
separate experiments (A). Phosphorylated tyrosine kinase
activity was analyzed by Western blotting and semi-quantified using
densitometry. Densitometry analysis revealed 2 prominent bands at 85 kDa (B) and 200 kDa (C). Data represent the mean
density reading from three separate Western blots corrected for
background ± S.E. and expressed as a percentage increase relative
to control uninfected cells at each time point (*, p < 0.01 5 min compared with 0 min; **, p < 0.05 at 5 min
compared with 15 and 30 min).
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Fig. 7.
Effect of genistein (100 µM) on the HRV-14 induction of mICAM-1
protein expression on bronchial epithelial cells at 0-96 h.
mICAM-1 protein expression was measured using immunocytochemistry and
expressed as a Pox score. Data are mean ± S.E. of three separate
experiments (*, p < 0.001 compared with untreated
HRV-14-infected cells). HRV-infected cells are represented by
black bars and HRV-infected cells preincubated with
genistein are represented by gray bars.
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DISCUSSION |
In this present study, we have investigated the mechanisms driving
the regulation of the major rhinovirus group receptor, ICAM-1 at the
epithelial cell level during infection. As ICAM-1 can exist in two
distinct forms, which appear to have opposing influences on host cell
infectivity, we postulate that the dynamic inverse relationship between
the membranous and soluble ICAM-1 receptor types is a critical catalyst
in the pathogenesis and outcome of HRV infection.
We have demonstrated the presence of distinct mRNA transcripts
coding for membrane-bound and soluble ICAM-1 in airway epithelial cells. We have also shown that these two receptors may be regulated independently. Consistent with previous studies (3, 12-14), our
experiments demonstrated that rhinovirus infection of NHBE cells,
increased the expression of mICAM-1 at both the protein and gene level
peaking in this study at 8 and 24 h after virus inoculation,
respectively, and thereafter, remained elevated above comparative
uninfected control cells for up to 96 h after inoculation.
In addition, we explored effects of HRV on sICAM-1 gene and protein
expression on the above cells. Rhinovirus appeared to down-regulate the
gene expression of sICAM-1 throughout the study period reaching
half-basal levels of expression by 24 h. This observed
down-regulation of sICAM-1 mRNA is supported by the absence of
detectable sICAM-1 in cell culture supernatants retrieved from HRV-infected cells. As virus and the antibody applied in the sICAM-1 ELISA utilize the same binding site on sICAM-1 molecules, the possibility of interference by HRV with the ELISA was eliminated by
assaying controls "spiked" with recombinant sICAM-1 (data not shown).
To our knowledge, we are the first to report the simultaneous
differential effect of HRV on both membrane and soluble forms of ICAM-1
receptor in an epithelial cell model, such that the virus appears to
induce the membrane-bound form, while dramatically decreasing the
soluble component; thereby facilitating and promoting cell infectivity.
Of particular interest, we have conducted other studies, which show
this same pattern of inverse relationship between mICAM-1 and sICAM-1
pattern of regulation following HRV inoculation of other epithelial
cell lines, BEAS-2B and H292 (data not shown) (27). However, these
studies also suggest that differences can occur in the time kinetics
and magnitude of responses dependent on cell type used. Indeed, this is
supported by the observations of Papi and Johnston (3), who observed
significant magnitude differences in viral-induced ICAM-1 cell surface
expression between primary bronchial epithelial cells and A549
(bronchial carcinoma-derived) epithelial cells. Thus direct comparison
of data between studies needs to take into account differences in cell
origin, culture techniques as well as assay conditions used, as all
these factors may account for observed differences in responsiveness to
HRV infection.
Taken together, these results suggest that sICAM-1 may be detrimental
to the virus as its competitive binding to available virus particles
would facilitate a defensive role in limiting viral infection of target
cells. Indeed, a recombinant form of sICAM-1 has been shown to have
inhibitory effects on HRV infection in vitro (17). Studies
in chimpanzees (18) and humans (19) have shown recombinant sICAM-1 to
have some prophylatic effects with reduced severity of experimental
rhinoviral colds. A recent in vivo study investigated
mICAM-1 expression on nasal scrape biopsies and sICAM-1 levels in nasal
lavage fluid from separate volunteer groups inoculated with
experimental rhinovirus (28). mICAM-1 expression was increased in 87%
of the volunteers following infection with rhinovirus; however,
sICAM-1 levels in the nasal lavage fluid were only increased in
47% of volunteers. This differs from our current study; we found no
detectable sICAM-1 in cell culture supernatants from HRV-infected
cells. This apparent discrepancy in results may be caused by
fundamental differences in the study design as our study is an in
vitro study using cell lines. In addition, as less than half the
volunteers exhibited an increase in sICAM-1 in the nasal lavage fluid,
it is equally possible that the sICAM-1 measured may be due to mICAM-1
dislodged from the cell surface as a result of the sampling process and
not through direct release. Time kinetics of ICAM-1 induction was
consistent with our study in that up-regulation of ICAM-1 occurred
within 24 h of infection and declined by day 5 (28).
Levels of infectious virus in cell culture supernatants from infected
cells increased 8-h postinoculation and remained elevated for up to
96 h, suggesting an increase in viral replication over time. These
data were confirmed by RT-PCR, in which a time-dependent increase in HRV RNA within NHBE cells was observed.
Blocking viral binding and subsequent viral internalization using
monoclonal antibodies against ICAM-1 resulted in the inhibition of both
the HRV-induced increase in mICAM-1 and down-regulation of sICAM-1
expression. In addition, viral titers in retrieved cell culture
supernatants were significantly lower in cells pretreated with ICAM-1
monoclonal antibodies, suggesting a reduction of initial viral binding,
entry, and subsequent infection. These data suggest that the observed
HRV effects on ICAM-1 expression are due to virus specific-epithelial
cell receptor interactions. This information may facilitate the design
of potential small molecule therapeutic inhibitors targeting HRV-cell
receptor interactions or subsequent intracellular events following
viral binding and release of genetic material into the cell.
The HRV-induced increase in mICAM-1 expression was inhibited by
cycloheximide indicating that HRV initiates de novo protein synthesis of ICAM-1. However, cycloheximide had no effect on sICAM-1 release from HRV-inoculated cells. Treatment of NHBE cells with actinomycin D also inhibited the HRV-induced increase in mICAM-1 suggesting HRV initiates transcription of the ICAM-1 gene.
In contrast, actinomycin D increased the release of sICAM-1 from HRV-inoculated NHBE cells, suggesting HRV may increase transcription of
a suppressor/accessory protein preventing the activity of a proteolytic
enzyme involved in the cleavage of mICAM-1 from the cell surface.
Possible candidate enzymes involved in the cleavage of mICAM-1 include
the metalloproteinases (MMPs), which are tightly regulated by tissue
inhibitors of metalloproteinases (TIMPs). In human gastric
adenocarcinoma cells, Helicobateur pylori has been
demonstrated to modulate MMP and TIMP secretion and that host MMP-3 and
a TIMP-3 homolog expressed by H. pylori mediate at least in
part of the host cell response to infection (29). A similar mechanism
may operate in HRV-infected bronchial epithelial cells. In addition, a
study utilizing astrocytes demonstrated that the mechanism involved in
sICAM-1 release was sensitive to metalloproteinase inhibitors (30).
Proteolytic cleavage of mICAM-1 has also been observed in
keratinocytes, where the addition of protease inhibitors resulted in a
dose-dependent inhibition of sICAM-1 production (31). It is
therefore plausible that HRV may modulate the release of sICAM-1 by
both down-regulating the gene expression of sICAM-1 and manipulating
the potential enzymatic reactions involved in the cleavage of mICAM-1
from bronchial epithelial cells. Further studies need to identify
enzymatic pathways responsible for the cleavage of mICAM-1 in bronchial
epithelial cells.
HRV has been shown to inhibit nuclear import, thus preventing signal
transduction into the nucleus (32). This could serve as one pathway
utilized by HRV during the down-regulation of sICAM-1 expression.
Alternatively, expression of sICAM-1 protein may be blocked at the
level of translation. Previous studies have demonstrated that certain
viruses may block translation of mRNA at the initiation step (33).
Indeed, a study conducted by Svitkin et al. (34) demonstrated that HRV inhibits host cell protein synthesis by cleaving
the eukaryotic initiation factors eIF4G11 and eIF4G1 resulting in a
60% decline in host protein synthesis by 6 h. This mechanism may
contribute to the HRV-induced inhibition of sICAM-1 release as no
sICAM-1 protein was detected at 0 h (8 h after inoculation with
HRV-14). Translation termination factors may serve as a target for the
virus. It has been demonstrated that certain isolated RNAs have an
affinity for eukaryotic translation termination factors, eRF1, and
eRF1·eRF3 complexes; to which they not only bind but also inhibit
eFR1-mediated release of protein precursor chains from ribosomes
(35).
Furthermore, HRV could modulate sICAM-1 secretion. Studies have
demonstrated that poliovirus, also a member of the picornaviridae family, inhibits the transport of both plasma and secretory proteins from the endoplasmic reticulum to the Golgi apparatus early in the
infection cycle (36-37). It is therefore plausible that the above
pathways, either solely or partly in combination could drive the
observed HRV-induced down-regulation in sICAM-1 release.
Since our studies have shown an HRV-induced increase in mICAM-1
expression at the transcriptional level, we proceeded to investigate the potential molecular mechanisms involved. Previous studies have
shown that HRV induced up-regulation of mICAM-1 gene
promoter activity involves initiation of NF
B proteins binding to the
NF
B binding site on the ICAM-1 gene promoter region (3).
Other studies have also indicated that tyrosine kinases may play a role in the regulation of the ICAM-1 gene (26, 38-39). We have
examined this intracellular pathway further and demonstrated that HRV
initiates rapid onset of tyrosine phosphorylation of multiple
substrates. There was strong phosphorylation of two substrates, 85 and
200 kDa, 5 and 15 min post-HRV inoculation. This response was totally inhibited by genistein at concentrations of 50 and 100 µM. Kelley and Drumm (40) also demonstrated that ICAM-1
expression was mediated through tyrosine kinases of 85 and 154 kDa in
endotheial cells and showed that ICAM-1 expression was completely
inhibited with genistein. Studies investigating tyrosine
phosphorylation events during Coxsackie virus, also a member of the
Picornaviridae family have also demonstrated an increase in tyrosine
phosphorylation of a 200-kDa protein (41). Huber et al. (41)
concluded that this protein was of cellular origin and may play a
critical role in effective viral replication. In addition, tumor
necrosis factor (TNF)-induced ICAM-1 expression has been demonstrated
to involve the tyrosine phosphorylation of an 85-kDa protein, which was
thought to be a cytoskeletal protein (41). Further studies utilizing more specific inhibitors for example herbimycin A, a selective inhibitor of Src-like kinases or tyrphostin, an inhibitor of Janus kinase (JAK) are required to identify these proteins. In addition, specific monoclonal antibodies to proteins of a similar molecular weight may be utilized to identify these substrates. In this study, genistein (100 µM) significantly inhibited the
HRV-induced up-regulation in mICAM-1 expression at 8 h with levels
decreasing to basal levels of expression by 96 h. These data
support the hypothesis that HRV may modulate mICAM-1 expression through
a tyrosine kinase-dependent signaling pathway.
In conclusion, we have demonstrated that HRV manipulates the
expression of both ICAM-1 receptors in airway epithelial cells to
promote and sustain infection. We have attempted to elucidate the
complex molecular mechanisms of ICAM-1 regulation and identified protein tyrosine kinases as critical components. It is plausible that
detailed dissection of the molecular driving forces involved in
coordinating the inverse relationship between membranous and soluble
ICAM-1 receptors in the context of HRV-epithelial cell membrane
interaction may lead to the development of novel anti-HRV therapeutic strategies.