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J Biol Chem, Vol. 274, Issue 14, 9707-9720, April 2, 1999
Rhinovirus Infection Induces Expression of Its Own Receptor
Intercellular Adhesion Molecule 1 (ICAM-1) via Increased
NF- B-mediated Transcription*
Alberto
Papi and
Sebastian L.
Johnston
From the University Medicine, University of Southampton,
Southampton General Hospital, Tremona Road,
Southampton SO16 6YD, United Kingdom
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ABSTRACT |
Virus infections, the majority of which are
rhinovirus infections, are the major cause of asthma exacerbations.
Treatment is unsatisfactory, and the pathogenesis unclear. Lower airway lymphocyte and eosinophil recruitment and activation are strongly implicated, but the mechanisms regulating these processes are unknown.
Intercellular adhesion molecule-1 (ICAM-1) has a central role in
inflammatory cell recruitment to the airways in asthma and is the
cellular receptor for 90% of rhinoviruses. We hypothesized that
rhinovirus infection of lower airway epithelium might induce ICAM-1
expression, promoting both inflammatory cell infiltration and
rhinovirus infection. We therefore investigated the effect of
rhinovirus infection on respiratory epithelial cell ICAM-1 expression
and regulation to identify new targets for treatment of virus-induced
asthma exacerbations. We observed that rhinovirus infection of primary
bronchial epithelial cells and the A549 respiratory epithelial cell
line increased ICAM-1 cell surface expression over 12- and 3-fold,
respectively. We then investigated the mechanisms of this
induction in A549 cells and observed rhinovirus-induction of ICAM-1
promoter activity and ICAM-1 mRNA transcription. Rhinovirus induction of ICAM-1 promoter activity was critically dependent upon up-regulation of NF- B proteins binding to the  187/178 NF-B binding site on the ICAM-1 promoter. The principal components of the rhinovirus-induced binding proteins were NF-B p65 homo- or
heterodimers. These studies identify ICAM-1 and NF-B as new targets
for the development of therapeutic interventions for virus-induced asthma exacerbations.
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INTRODUCTION |
Asthma is increasingly common and now affects up to 30% of the
population in westernized countries (1). Asthma exacerbations are the
major cause of asthma morbidity and mortality. Respiratory viral
infections have recently been associated with the majority of asthma
exacerbations in both adults and children. In community-based studies,
viral infections were identified in 80-85% of exacerbations in
children and 44% of exacerbations in adults (2, 3). Viral infections
have also been strongly implicated in more severe asthma exacerbations
requiring hospitalization in both children and in adults (4). In all of
these studies, rhinovirus infections caused around 65% of
exacerbations in which a virus was identified (2-4).
Rhinovirus-induced asthma exacerbations therefore cause enormous
morbidity, especially among children, and represent a major health and
economic problem.
To date, no safe effective therapy is available, since prophylactic
inhaled steroids are ineffective (5), while intervention with high dose
inhaled steroids is only partially effective (6). A better
understanding of the mechanisms involved in rhinovirus-induced asthma
exacerbations would greatly aid the development of new therapies for
this common condition.
The mechanisms by which rhinoviruses trigger asthma exacerbations are
poorly understood. Asthma is an inflammatory disease of the lower
respiratory airways, and lower airway inflammatory changes have been
described during experimental rhinovirus colds. A marked bronchial
CD3+, CD4+, and CD8+ T lymphocyte
and eosinophil infiltration was observed in biopsies taken at the
height of cold symptoms in both normal and in asthmatic subjects (7).
However, the eosinophil infiltrate was more prolonged in the asthmatic
subjects, still present 6-8 weeks after infection, while the
eosinophil counts in normal subjects had returned to base line (7).
Rhinovirus experimental infections have also been reported to increase
allergen-induced eosinophil numbers in bronchial lavage fluid in atopic
rhinitic subjects, while no change in eosinophil numbers was observed
in normal subjects (8), and to increase eosinophil products in sputum
supernatants in asthmatic subjects (9). These data combined strongly
suggest that rhinovirus-induced bronchial lymphocyte and eosinophil
infiltration and activation are probably very important mechanisms in
virus-induced asthma exacerbations.
The increased airway reactivity demonstrated in experimental rhinovirus
infections in asthmatic subjects (9, 10) and atopic subjects (11) and
the induction by rhinovirus of late asthmatic responses to inhaled
allergen (8, 11) also provide indirect evidence of a link between lower
respiratory inflammation during rhinovirus experimental infections and
the mechanisms of virus-induced asthma exacerbations. Finally, evidence
that the lymphocytic and eosinophilic inflammation observed during
rhinovirus experimental infections (7-9) is probably also an important
mechanism involved in virus-induced asthma exacerbations comes from the fact that asthma exacerbations have been induced by experimental rhinovirus infections (9, 12).
Rhinovirus RNA has recently been detected in bronchial lavage cells
taken during experimentally induced colds, suggesting that rhinovirus
can promote local inflammation by direct infection of the lower airways
(13). Indeed, rhinoviruses are capable of prolonged, noncytolytic
infection of respiratory epithelial cells and induce production of
proinflammatory cytokines such as
IL-6,1 IL-8, and
granulocyte-macrophage colony-stimulating factor (14-17). Taken
together, these data suggest that lower airway rhinovirus-induced inflammatory cell recruitment is a critical event in rhinovirus-induced asthma exacerbations.
Intercellular adhesion molecule-1 (ICAM-1) is a cell surface
glycoprotein belonging to the immunoglobulin supergene family that is
involved in leukocyte trafficking and accumulation at sites of
inflammation. In addition to modulating eosinophil infiltration by
binding to its ligand CD18/CD11b, ICAM-1 is involved in lymphocyte infiltration by binding to CD18/CD11a expressed on both
CD4+ and CD8+ T lymphocytes. There is now
considerable evidence that ICAM-1 plays a central role in recruitment
and activation of these inflammatory cells in the pathogenesis of
asthma. Epithelial ICAM-1 expression is increased by allergen challenge
in both allergic rhinitis and conjunctivitis (18-20). High levels of
epithelial ICAM-1, along with intraepithelial inflammatory cell
infiltration, have been described in bronchial biopsies from subjects
both with stable asthma and after allergen challenge (21, 22). Direct
evidence for the important role of ICAM-1 in eosinophil recruitment
came from a primate study that demonstrated that allergen challenge causing a dual asthmatic response up-regulated ICAM-1 expression in
airway epithelium and endothelium, that eosinophil infiltration into
the airways correlated with the epithelial ICAM-1 levels, and that
anti-ICAM-1 antibodies prevented both the eosinophil influx and airway
hyperreactivity (23). The critical importance of ICAM-1 in asthma
pathogenesis has been emphasized by two further recent studies in
murine models of asthma, which demonstrated ICAM-1 regulation of
lymphocyte and eosinophil recruitment to the lower airway (24, 25).
Since epithelial infiltration of each of these cell types is
specifically implicated in rhinovirus induced asthma (7-9), respiratory epithelial ICAM-1 expression is likely to play a very important role in inflammatory cell infiltration associated with rhinovirus-induced asthma exacerbations.
In addition to its important role in inflammatory cell recruitment and
activation, ICAM-1 may play a critical role in rhinovirus-induced asthma exacerbations, since it is also the cellular receptor for the
major group (90%) of rhinoviruses (26, 27). Having observed previously
that rhinoviruses are able to infect lower respiratory epithelial cells
for several days without causing cytopathic effect and able to induce
proinflammatory protein expression (16), we hypothesized that
rhinovirus infection of lower respiratory epithelial cells would also
induce increased expression of ICAM-1. Rhinovirus induction of ICAM-1
in lower respiratory epithelial cells could then not only promote
intraepithelial inflammatory cell recruitment and activation but also
increase the severity of the epithelial cell infection and therefore
further exacerbate the airway inflammation. Modulation of ICAM-1
expression would then be expected to have profound effects on the
epithelial inflammatory cell infiltration associated with
rhinovirus-induced asthma exacerbations.
To investigate this hypothesis, studies were undertaken to determine
whether rhinovirus infection has the ability to modulate respiratory
epithelial ICAM-1 expression in an in vitro model. Having
found marked rhinovirus-induced ICAM-1 up-regulation in both primary
bronchial epithelial cells and a human lower respiratory cell line, we
investigated the intracellular mechanisms of rhinovirus induction of
ICAM-1 expression to identify potential targets for modulation of
rhinovirus-induced ICAM-1 in the therapy of rhinovirus-induced asthma exacerbations.
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MATERIALS AND METHODS |
Cell Culture
A549 cells, a type II respiratory epithelial cell line, were
obtained from the American Type Culture Collection (ATCC; Rockville, MD), and Ohio HeLa cells were obtained from the Medical Research Council Common Cold Unit (Salisbury, UK). Cells were split weekly and
cultured at 37 °C in 5% carbon dioxide in Eagle's minimal essential medium supplemented with 4 mM
L-glutamine, 80 mg/ml of gentamycin, and 10% fetal bovine
serum (Sigma, Poole, UK). Primary human bronchial epithelial cells were
obtained by bronchial brushing from normal patients undergoing surgery.
Cells were removed from the brush by vigorous shaking and were
disaggregated in Clonetics (San Diego, CA) bronchial epithelial cell
medium containing 3 mM DTT for 15 min. After washing, cells
were plated onto collagen-coated 16-mm diameter culture wells and grown
to confluence in bronchial epithelial cell medium. Cells were passaged
in 100-mm Petri dishes and used in the assays at passages 3 and 4. For
experiments, 70% confluent cells were detached using 0.05% trypsin
with 0.02% EDTA and seeded at 2 × 105 cells/well in
12-well culture plates. These cells are >95% cytokeratin 18-immunoreactive epithelial cells as assessed by immunofluorescence microscopy.
Viral Stocks
Rhinovirus types 16, 9 (major group), and 2 (minor group) were
obtained from the Medical Research Council Common Cold Unit, and their
identity was confirmed by neutralization with specific antiserum
(ATCC). Viral stocks were generated by infecting monolayer cultures of
HeLa cells until cytopathic effects were fully developed. Cells and
supernatants were harvested, cells were disrupted by freezing and
thawing, cell debris was pelleted by low speed centrifugation, and the
resulting clarified supernatants were frozen at 70 °C.
Rhinovirus titration was performed on the frozen aliquots by exposing
confluent monolayers of HeLa cells in 96-well plates to serial 10-fold
dilutions of viral stock. Plates were cultured for 5 days in 4%
minimal essential medium at 37 °C in 5% CO2. Cytopathic
effect was assessed by visual assessment and by assessment of the
continuity of the monolayer after fixation in methanol and staining
with 0.1% crystal violet. Tissue culture infective dose 50%
(TCID50)/ml values were determined (28), and virus at a
multiplicity of infection (MOI) of 1 was used for all of the
experiments except where indicated.
Rhinovirus Inactivation
For selected experiments, inactivation/filtration of the virus
was performed by three different methods.
Prevention of Virus-Receptor Binding
Viruses were precoated with excess soluble receptor to saturate
the receptor binding sites on the virus capsid. Virus stock solutions
were preincubated with recombinant soluble ICAM-1 (sICAM; a gift of P. Esmon, Bayer Corp., Berkeley, CA) at a concentration of 1 mg/ml for 30 min at room temperature.
Prevention of Virus Replication
Viruses were inactivated by exposure to UV light at 1200 mJ/cm2 for 30 min.
Filtration of Virus from Inoculum
Virus particles were removed from inocula by ultrafiltration
through membranes (Amikon, London, UK) to remove all molecules greater
than 30 kDa, performed according to the manufacturer's instructions.
For each method, confirmation of complete inactivation was carried out
by microtiter plate assay for rhinovirus infectivity as described above.
Measurement of ICAM-1 Surface Protein Expression
2 × 105 A549 or primary bronchial epithelial
cells were cultured in 12-well plates. When confluent, virus at an MOI
of 1 or control media was added, and incubation continued for various periods of time between 1 and 72 h. Dose-response studies were carried out using 0.05, 0.1, 0.5, 1, and 2 MOI, and cells were harvested at 8 h. Similarly, the effects of inactivated/filtered virus and the effects on primary bronchial epithelial cells were studied at 8 h. At the desired time points, cells were detached intact by incubation with 0.5 ml/well cell dissociation solution (Sigma) at 37 °C for 10 min. More than 95% of cells were viable as
determined by trypan blue dye exclusion. 105 cells were
then washed and resuspended in PBA (phosphate-buffered saline, 1%
bovine serum albumin, 0.1% sodium azide) and incubated with saturating
amounts of fluorescein isothiocyanate-conjugated anti-human ICAM-1
(CD54) antibody or isotype-specific control antibody (Serotec, Oxford,
UK) for 30 min at 4 °C in the dark. After washing, 104
cells were analyzed for fluorescence by single color flow cytometry on
a FACScan analyzer (Becton Dickinson, San Jose, CA). Mean fluorescence intensity was measured and normalized relative to noninfected control
cells after subtraction of background staining.
ICAM-1 mRNA Analysis
5 × 106 A549 cells were cultured in 100-mm
plates until confluent, and medium alone or rhinovirus type 16 was
added for various times between 1 and 24 h. Studies with
inactivated/filtered virus were performed at 8 h. At the desired
time points, cells were harvested, and ICAM-1 mRNA expression was
evaluated by RT-PCR. Whole cell RNA was extracted using Trizol
according to the manufacturer's instructions (Life Technologies, Inc.,
Paisley, UK). One µg of total RNA was reverse transcribed by
superscript reverse transcriptase (100 units; Promega, Southampton, UK)
in a total volume of 10 µl at 37 °C for 1 h using P1 (24 ng/ml) as specific primer (Table I). The cDNA (2.5 µl) was
amplified by PCR in the presence of a master mix containing PCR buffer,
MgCl2 (1.5 mM), 1.25 units of Taq
DNA polymerase (Promega), 0.2 mM dNTPs, and 0.6 mM specific primer pair (P1 and P2; Table I). Cycling
conditions were 1 min at 94 °C, 1 min at 55 °C, and 2 min at
72 °C for 25 cycles. First round products were diluted 1:100, and
2.5 µl was thereafter used for a nested amplification under the same
PCR conditions, with P3 and P4 as inner primers (Table I). Final PCR
products (10 µl) were electrophoresed through 1.5% agarose gels,
stained in ethidium bromide, and photographed under UV light. In
parallel, mRNA for adenine phosphoribosyltransferase (APRT), using
primers indicated in Table I for 40 cycles at 56 °C, was evaluated in each sample as housekeeping gene
control. Densitometry was performed using a scanning densitometer, and
densitometric analysis was performed using the Phoretix program
(Biometra Ltd., Newcastle-upon-Tyne, UK) to express ICAM-1 mRNA
relative to APRT mRNA.
To confirm the quantitative nature of the PCR, cell lysate was diluted
3-fold in triplicate and subjected to RNA extraction, RT-PCR, and
densitometric analysis.
ICAM-1 Nuclear Transcription Analysis
Isolation of nuclei and in vitro nuclear
transcription were performed using standard procedures (29). Confluent
cell monolayers (2.5 × 107 cells) were incubated with
medium alone or rhinovirus 16 for 1 h. Cells were then washed
twice with phosphate-buffered saline, harvested, and centrifuged at
500 × g for 5 min. The cell pellet was resuspended in
1 ml of 10 mM Tris-HCl, pH 8.4, 1.5 mM
MgCl2, 0.14 M NaCl and lysed by the addition of
5% Nonidet P-40. The progress of lysis was monitored by trypan blue
exclusion. Nuclei were pelleted by centrifugation at 500 × g for 1 min and were washed twice in 20 mM
Tris-HCl, pH 8.3, 20% glycerol, 100 mM KCl, 4.5 mM MgCl2, 2 mM DTT.
In vitro nuclear transcription was carried out for 45 min at
30 °C in 200 ml of this buffer supplemented with 1 mM
each of ATP, GTP, CTP, and UTP.
For each sample, total RNA was extracted from 107 nuclei,
before and after in vitro transcription, in the presence or
absence of an RNA polymerase II inhibitor  -amanitin (1 mg/ml) (30). ICAM-1 RT-PCR was subsequently performed (see above) for each different
condition to detect in vitro transcribed products.
Reporter Gene Constructs
The ICAM-1 promoter-chloramphenicol acetyltransferase (CAT)
constructs were a generous gift of Dr. K. Degitz (Ludwig-Maximilians University, Munchen). They contained sequential deletions (1160, 277, 182, 135, 88) of the ICAM-1 5'-flanking region linked to
the coding region of the CAT reporter gene (31). A plasmid containing
the longest promoter deletion with a mutated 187/178 NF-B
sequence (1160m; mutated from TGGAAATTCC to TctAgATTag and confirmed
by sequencing) and pBRAMScat2 vectors, composed of the CAT reporter
gene and the herpes simplex virus minimal thymidine kinase promoter
alone or linked to fragments of the ICAM-1 promoter (199/170 or
199/182), were also kindly provided (31, 32).
Cell Transfection and CAT Assay
A549 cells were transfected with reporter constructs (10 µg)
at 80% confluency by the calcium phosphate precipitation technique for
5 h, glycerol-shocked with 1× HeBS (0.02 M Hepes,
0.135 M NaCl, 0.5 mM
Na2HPO4, 5.5 mM
D-glucose, pH 7.1), 15% glycerol for 30 s and washed.
Transfected cells were cultured in 10% minimal essential medium for
24 h, and rhinovirus 16 or medium alone was added. At designated
time points, cells were harvested, and cell extracts were prepared by
three cycles of rapid freeze-thawing in 0.25 M Tris, pH
8.0. Each sample was incubated at 65 °C for 15 min to inactivate
endogenous transacetylases. Protein content was determined
photometrically using the Bio-Rad protein assay (Bio-Rad).
Protein-equivalent aliquots were assayed for CAT activity according to
standard protocols (33). The assay was performed at 37 °C for 60 min
in a reaction mixture of 1 mM acetyl coenzyme A (Amersham
Pharmacia Biotech) and 0.1 µCi of 14C-chloramphenicol.
Acetylated and unacetylated forms were resolved by thin layer
chromatography, visualized by autoradiography, and measured on a
scintillation counter. CAT activity was expressed as percentage of
chloramphenicol converted to its acetylated derivatives.
Electrophoretic Mobility Shift Assay (EMSA)
Preparation of Nuclear Extracts--
Uninfected and
rhinovirus-infected A549 cells were prepared as described previously.
At the desired time points (0, 30, 60, 90, and 120 min), the cells were
mechanically detached, and nuclear extracts were obtained by a
modification of the the method of Dignam et al. (34).
Briefly, after washing with phosphate-buffered saline, cells were
centrifuged at 4 °C and resuspended in buffer A (10 mM
Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM
KCl, 0.5 mM DTT) with freshly added protease inhibitors (10 mg/ml leupeptin, 5 mg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride). Membrane lysis was achieved by adding
0.5% Nonidet P-40 followed by vigorous agitation and incubation on ice
for 5 min. The nuclei were then pelleted at 4 °C and resuspended in
buffer C (20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM EDTA, 0.5 mM DTT,
and freshly added protease inhibitors as above). This suspension was
incubated on ice for 15 min and centrifuged. The protein concentration
of the nuclear extracts was photometrically determined using the
Bio-Rad protein assay.
Oligonucleotide Probes (Table
II)--
Double-stranded
oligonucleotides containing wild-type and mutated sequences of ICAM-1
AP-1, NF-B, Sp1, and C/EBP recognition sequences were obtained
commercially (Oswell DNA Service, Southampton, UK). Mutant sequences
were identical to those used in the mutant reporter constructs. Probes
containing NF-B, AP-1, or Sp1 consensus sequences were commercially
obtained (Promega).
Oligonucleotides were end-labeled with [ -32P]ATP and
T4 polynucleotide kinase (Promega). Equal amounts (5 µg) of nuclear
protein were incubated with 10 fmol of probe in binding buffer (10 mM Tris, pH 7.5, 5% glycerol, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 1.25 µg of poly(dI-dC)·poly(dI-dC)) for 30 min at room temperature. Complexes were resolved on 5% nondenaturing polyacrylamide gels in TBE buffer (50 mM Tris, pH 8.0, 50 mM boric acid, 1 mM EDTA) containing 4%
glycerol. Electrophoresis was performed at 10 V/cm for 2-3 h. Gels
were dried, and binding was assessed by autoradiography.
Supershift EMSA--
Supershift assays were used to study which
members of the NF-B family were involved in rhinovirus-induced
formation of complexes with the ICAM-1 promoter sequence 199/170.
One µl of rabbit polyclonal antibodies against each of p65, p50, p52,
c-Rel, and Rel-B (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were
added to 2 µg of nuclear extracts for 15 min at 4 °C before the
incubation with radiolabeled probe as described above. Rabbit preimmune
serum was used as negative control.
Statistical Analysis
Data were expressed as mean ± S.E., and comparison between
groups was performed by analysis of variance for multiple comparisons and by paired Student's t tests for individual comparisons.
All experiments were carried out at least three times.
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RESULTS |
Rhinovirus Induces ICAM-1 Cell Surface Protein Expression in A549
Cells and in Primary Bronchial Epithelial Cells--
Preliminary
studies indicated that rhinovirus infection of A549 cells up-regulated
ICAM-1 surface expression at 8 h postinoculation. Dose-response
studies in A549 cells exposed to rhinovirus 16 were therefore carried
out to determine if the induction of ICAM-1 occurred in a dose-response
manner. Cell surface ICAM-1 expression was studied by flow cytometry
8 h after infection; enhanced expression of ICAM-1 relative to
uninfected cells was observed at 0.1 TCID50/cell and peaked
at 1 TCID50/cell, where there was 3.5-fold induction over
uninfected cell levels of ICAM-1 expression (Fig.
1). Based on these dose-response data, a
MOI of 1 TCID50/cell was utilized in all subsequent
studies.
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Fig. 1.
Dose response of rhinovirus-induced ICAM-1
surface expression on A549 respiratory epithelial cells. Surface
ICAM-1 expression was measured by flow cytometry on A549 epithelial
cells cultured for 8 h with medium alone (control) or rhinovirus
16 at titers ranging from 0.05 to 2 MOI. ICAM-1 induction by rhinovirus
infection is expressed as -fold increase over control uninfected cells.
Data are mean ± S.E. of at least four separate experiments (**,
p < 0.001 compared with control). Rhinovirus infection
of A549 respiratory epithelial cells induced a significant
up-regulation of ICAM-1 surface expression at multiplicities of
infection of 0.5, 1, and 2, with peak induction occurring at an MOI of
1.
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To evaluate the temporal kinetics of ICAM-1 induction by rhinovirus,
surface ICAM-1 expression was studied at 0, 1, 4, 8, 16, 24, 48, and
72 h post-rhinovirus 16 infection. Significant up-regulation was
apparent within 4 h, was maximal at 8 h, and was still
significantly increased at 48 and 72 h after infection (Fig.
2). The levels of rhinovirus-induced
ICAM-1 were similar in magnitude to those observed with interferon-
(10 units/ml) treatment, which was used as a positive control (data not
shown). In view of the time course results, an 8-h infection was chosen for comparative studies to investigate the receptor specificity and
virus specificity of the up-regulation.
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Fig. 2.
Time course of rhinovirus-induced ICAM-1
surface expression on A549 respiratory epithelial cells. Surface
ICAM-1 expression was measured by flow cytometry on A549 epithelial
cells cultured with medium alone (control) or rhinovirus 16 at an MOI
of 1 for 1, 4, 8, 16, 24, 48, and 72 h. ICAM-1 induction by
rhinovirus infection is expressed as -fold increase over control
uninfected cells. Data are mean ± S.E. of at least four separate
experiments (*, p < 0.01; **, p < 0.001 compared with control). Rhinovirus infection of A549 respiratory
epithelial cells induced a significant up-regulation of ICAM-1 surface
expression within 4 h after inoculation, which peaked at 8 h
after infection and was still evident up to 72 h.
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Having investigated rhinovirus regulation of ICAM-1 expression in the
human lung carcinoma epithelial cell line A549, we wished to determine
whether the same effects could be observed in primary human bronchial
epithelial cells. The effect of rhinovirus infection on respiratory
epithelial ICAM-1 surface expression was studied by flow cytometry in
primary human bronchial epithelial cells. We observed that rhinovirus
infection for 8 h increased ICAM-1 expression 12.7 times the
values of control sham-infected cells (Fig.
3). These data confirmed that rhinovirus
infection of both A549 cells and primary bronchial epithelial cells
were associated with markedly increased ICAM-1 surface expression. We
therefore investigated the mechanisms of this induction in A549 cells,
since the numbers of cells required for subsequent experiments
precluded the use of primary cells.
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Fig. 3.
Rhinovirus-induced ICAM-1 surface expression
on primary bronchial epithelial cells. Surface ICAM-1 expression
was measured by flow cytometry on primary bronchial epithelial cells
cultured with medium alone (control) or rhinovirus 16 at an MOI of 1 for 8 h. ICAM-1 induction by rhinovirus infection is expressed as
-fold increase over control uninfected cells. Data are mean ± S.E. of four separate experiments (***, p < 0.0001 compared with control). Rhinovirus infection of primary bronchial
epithelial cells induced a marked up-regulation of ICAM-1 surface
expression 8 h after inoculation.
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The Effects of Rhinovirus Replication and Receptor Binding on
Rhinovirus-induced ICAM-1 Cell Surface Expression--
Since the virus
inoculum was a crude preparation, we wished to confirm that the
induction of ICAM-1 surface expression was a result of virus-specific
effects rather than a result of stimulation by other soluble products
such as cytokines present in the inoculum. We were also interested in
investigating whether any rhinovirus-specific effect observed was a
result of virus replication or of virus-receptor binding. We therefore
elected to inactivate rhinovirus by two methods: UV inactivation to
prevent replication but not receptor binding and precoating with
soluble receptor (sICAM) to prevent receptor binding. Finally we
filtered the inoculum through a molecular weight filter to remove all
virus particles and RNA but not small molecules such as cytokines.
As can be observed in Fig. 4, incubation
with UV-inactivated virus resulted in marked inhibition of
rhinovirus-induced ICAM-1 surface protein expression (from 3.49 ± 0.3- to 1.63 ± 0.1-fold induction); however, there was still a
small but significant induction with UV-inactivated rhinovirus over
control cells (p < 0.05). In contrast, sICAM
inactivated and filtered virus completely abrogated the induction
observed with rhinovirus (Fig. 4). These results suggest that
approximately one-quarter of the ICAM-1 up-regulation observed with
live rhinovirus occurs independently of viral replication, as a result
of virus-receptor interaction, while the major part is dependent upon
viral replication.
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Fig. 4.
Effect of inactivation of virus replication
and prevention of virus-receptor binding on rhinovirus induction of
ICAM-1 surface expression on A549 respiratory epithelial cells and
evaluation of serotype and receptor type specificity. Surface
ICAM-1 expression was measured by flow cytometry on A549 epithelial
cells cultured for 8 h under the following conditions: medium
alone (control); live rhinovirus 16 at an MOI of 1 (RV16);
UV-inactivated rhinovirus 16 (UV RV16); sICAM-1-pretreated
rhinovirus 16 (sICAM RV16); rhinovirus 16 physically removed
by filtration (Filtered RV16); live rhinovirus 9 at an MOI
of 1 (RV9); live rhinovirus 2 at an MOI of 1 (RV2); and sICAM-pretreated rhinovirus 2 (sICAM-1
RV2). ICAM-1 induction by rhinovirus infection is expressed as
-fold increase over control uninfected cells. Data are mean ± S.E. of at least four separate experiments (*, p < 0.01; **, p < 0.001 compared with control). Live
rhinovirus type 16 (major group, using ICAM-1 as virus receptor)
induced a greater than 3-fold increase in ICAM-1 surface expression
compared with control uninfected cells. Inactivation by physical
removal of virus particles (Filtered RV16) and prevention of
virus-receptor binding (sICAM RV16) completely abrogated
rhinovirus induction of ICAM-1 expression, while prevention of viral
replication (UV RV16) only partly inhibited rhinovirus-induced ICAM-1
expression. Other rhinovirus serotypes, rhinovirus 9 (major group) and
rhinovirus 2 (minor group), were equally able to up-regulate surface
ICAM-1 expression, while pretreatment of rhinovirus 2 (which does not
use ICAM-1 as virus receptor) with irrelevant soluble receptor
(sICAM-1 RV2) had no effect on rhinovirus induction of
ICAM-1 expression.
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Rhinovirus Induction of ICAM-1 Is Not Virus
Receptor/Strain-specific--
The major group (90%) of rhinoviruses
use ICAM-1 as their cell surface receptor (26, 27), while the remainder
(minor group) use members of the low density lipoprotein receptor
family (35). Having observed rhinovirus induction of ICAM-1 expression
with a major group rhinovirus, rhinovirus 16, we wished to investigate whether rhinovirus induction of ICAM-1 up-regulation is strain- or
group (receptor)-restricted.
We therefore compared the stimulatory effect of rhinovirus 16, rhinovirus 9 (both major group), and rhinovirus 2 (minor group), all at
an MOI of 1, on A549 cell ICAM-1 surface expression at 8 h
postinfection. As shown in Fig. 3, rhinovirus 16, rhinovirus 9, and
rhinovirus 2 were equally effective at increasing ICAM-1 surface
expression, demonstrating that rhinovirus-induced ICAM-1 up-regulation
occurs with at least three of the many different rhinovirus serotypes
and that the induction was not receptor-restricted. Furthermore,
pretreatment of rhinovirus 2 with sICAM did not alter the ability of
this minor group rhinovirus to induce ICAM-1. Having observed that
sICAM pretreatment completely abolished (Fig. 4) the ICAM-1 expression
induced by the major group rhinovirus, rhinovirus 16, these findings
support our interpretations of the preceding data relating to the
respective contributions of virus-receptor binding and virus
replication to ICAM-1 induction by rhinoviruses.
Induction of ICAM-1 mRNA in A549 Cells by Live and Inactivated
Rhinovirus--
Having found rhinovirus-induced increases in ICAM-1
epithelial cell surface protein expression, we wished to test the
effects of rhinovirus infection on epithelial cell ICAM-1 mRNA expression.
First, we wished to determine that the PCR analysis was quantitative
over the range of input RNA used in the study. To investigate this, a
cell lysate known to produce a strong band upon PCR was serially
diluted 3-fold and subjected to extraction, RT-PCR, and densitometric
analysis in triplicate. These studies clearly demonstrated that the
ICAM-1 RT-PCR used in the subsequent studies was quantitative in a
linear fashion (Fig. 5).
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Fig. 5.
RT-PCR for ICAM-1 mRNA expression in A549
respiratory epithelial cells is quantitative. Rhinovirus-infected
cell lysate was serially diluted 3-fold and subjected to extraction,
RT-PCR, and densitometric analysis. Each point represents the mean ± S.E. optical density of three separate extractions/RT-PCRs. There is
a linear relationship between the degree of dilution of input template
RNA and the optical density, indicating that the ICAM-1 RT-PCR used in
the subsequent studies was quantitative.
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The time course of ICAM-1 mRNA induction in response to rhinovirus
16 was studied by RT-PCR at 0, 1, 3, 6, 8, 12, 16, and 24 h after
rhinovirus infection. A549 cells incubated with medium alone did not
contain detectable levels of ICAM-1 mRNA (Fig.
6). In accordance with our findings on
surface expression, a consistent response to rhinovirus infection was
noted, with clear time-dependent increases in ICAM-1
mRNA expression being induced by rhinovirus infection (Table
III). A representative experiment is
depicted in Fig. 6, where an early increase in levels of ICAM-1
mRNA was detectable at 1 h, peaked at 8 h, and reduced
toward but not as far as base line up to 24 h.
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Fig. 6.
Time course of rhinovirus-induced ICAM-1
mRNA expression in A549 respiratory epithelial cells.
Representative (of at least three separate experiments) RT-PCR analysis
for ICAM-1 and APRT (housekeeping gene) expression in A549 respiratory
epithelial cells uninfected (0 h) and incubated with rhinovirus 16 at
an MOI of 1 for the indicated times are shown. Above are
ethidium bromide-stained gel electrophoreses of products of RT-PCR for
APRT and ICAM-1, and below are the ICAM-1/APRT ratios
determined by densitometric analysis. Rhinovirus infection of A549
respiratory epithelial cells induced increased ICAM-1 mRNA
expression, which was just detectable at 1 h, peaked at 8 h,
and was still clearly evident at 24 h after inoculation.
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Also consistent with the cell surface expression, UV-inactivated virus
(Fig. 7, lane 3)
resulted in a marked but incomplete inhibition of ICAM-1 mRNA
induction compared with live virus (Fig. 7, lane
2), whereas sICAM pretreatment (Fig. 7, lane
4) or filtration (Fig. 7, lane 5) of
the virus completely abrogated the response (Table
IV).
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Fig. 7.
Effect of inactivation of virus replication
and prevention of virus-receptor binding upon rhinovirus induction of
ICAM-1 mRNA expression in A549 respiratory epithelial cells.
Representative (of at least three separate experiments) RT-PCR analysis
for ICAM-1 and APRT (housekeeping gene) expression in A549 respiratory
epithelial cells uninfected (lane 1) or incubated
for 8 h with live rhinovirus 16 (lane 2),
UV-inactivated rhinovirus 16 (lane 3), rhinovirus
16 precoated with sICAM-1 (lane 4), or rhinovirus
16 removed by filtration (lane 5) is shown.
Above are ethidium bromide-stained gel electrophoreses of
products of RT-PCR for APRT and ICAM-1, and below are the
ICAM-1/APRT ratios determined by densitometric analysis. Rhinovirus
induction of ICAM-1 mRNA expression (lane 2)
was completely abolished by prevention of virus-receptor binding by
sICAM-1 precoating and by filtration (lanes 4 and
5) and was partly reduced by prevention of viral replication
by UV inactivation (lane 3).
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Rhinovirus Infection of A549 Cells Up-regulates ICAM-1 Gene
Transcription--
To determine whether the observed increases in
ICAM-1 mRNA and protein expression in response to rhinovirus
infection of A549 cells were mediated by increased ICAM-1 gene
transcription, de novo synthesis of ICAM-1 mRNA (nuclear
run-off) was studied in nuclei obtained from A549 cells after a 1-h
rhinovirus infection and in control noninfected cells.
In accordance with the observed mRNA time course studies, ICAM-1
mRNA was undetectable in nuclei from control noninfected cells,
either before (Fig. 8, lane
1) or after (Fig. 8, lane 2) in
vitro transcription, while a weak band of ICAM-1 mRNA was
detectable after a 1-h rhinovirus 16 infection but without in
vitro transcription (Fig. 8, lane 3). The
amount of ICAM-1 mRNA was markedly increased by 45-min in
vitro transcription (Fig. 8, lane 4),
indicating that rhinovirus infection of A549 cells resulted in
increased de novo ICAM-1 mRNA transcription (Table
V). This was confirmed by the fact that
the rhinovirus-induced increase in ICAM-1 mRNA observed during
in vitro transcription was abolished in the presence of
-amanitin, a DNA-dependent RNA polymerase II inhibitor
(30), (Fig. 8, lane 5; Table V). From these
results, we concluded that rhinovirus infection of A549 cells induces a
rapid increase in ICAM-1 gene transcription.
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Fig. 8.
Rhinovirus induction of de novo
ICAM-1 gene transcription in A549 respiratory epithelial
cells. Nuclei from uninfected or rhinovirus-infected A549 cells
were used for a nuclear in vitro transcription assay.
Representative (of at least three separate experiments) RT-PCRs for
ICAM-1 and APRT (housekeeping gene) were performed on nuclear RNA
obtained from A549 cells incubated in each of the following conditions:
uninfected control cells before (lane 1) and
after (lane 2) in vitro transcription;
rhinovirus 16-infected cells (1 h at an MOI of 1) before
(lane 3) and after in vitro
transcription (lane 4); and cells after in
vitro transcription in the presence of -amanitin
(lane 5). No ICAM-1 mRNA was detectable in
control uninfected cells, either before or after in vitro
transcription (lanes 1 and 2,
respectively). Increased expression of ICAM-1 mRNA was just
detectable after 1-h rhinovirus infection but before in
vitro transcription (lane 3). ICAM-1
mRNA expression was markedly increased by 45-min in
vitro transcription (lane 4), this increase
is completely inhibited by the addition of the RNA polymerase II
inhibitor, -amanitin (lane 5).
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Rhinovirus Infection of A549 Cells Increases ICAM-1 Promoter
Activity--
Having demonstrated rhinovirus induction of ICAM-1 gene
transcription, we then carried out studies to determine whether
rhinovirus infection of A549 cells increased ICAM-1 promoter activity.
A549 cells were transiently transfected with constructs containing the
CAT reporter gene, whose transcription was regulated by portions of the
ICAM-1 promoter. Time course experiments with a construct containing
the full-length promoter (1160 bp) showed that rhinovirus 16-infected
cells had significantly increased ICAM-1 promoter activity compared
with control cells at all of the tested time points (24, 48, 72, 96, 120 h, data not shown). At 24 h of infection, induction of
ICAM-1 promoter activity was maximal, promoter activity being barely
detected in control cells (acetylation 2 ± 0.7%), while it was
markedly increased in the rhinovirus-infected cells (acetylation
31.9 ± 9%, p < 0.01). The 24-h time point was
therefore utilized in subsequent experiments to investigate rhinovirus
induction of shorter deletions of the ICAM-1 promoter, to identify the
precise sites in the promoter induced by rhinovirus infection.
Rhinovirus Infection Induces Multiple Transcription Factors Binding
to the ICAM-1 Promoter--
Sequence analysis of the proximal ICAM-1
promoter has revealed potential binding sites for several transcription
factors including AP-1 (284/278), Sp1 (206/201), C/EBP
(199/196), and NF-B (187/178) and (62/53), which also
overlaps with a further Sp1 binding motif (59/54) (36-39). To
further understand the mechanisms by which rhinovirus induces ICAM-1
promoter activity, studies were undertaken to investigate whether
rhinovirus infection of A549 cells could increase the binding activity
of relevant transcription factors in nuclei extracted from infected and
uninfected lung epithelial cells, using labeled probes containing each
of the potential binding sites in EMSAs.
199 to 170 Probe Containing C/EBP (199/196) and NF-B
(187/178) Sites--
Two retarded complexes were observed using
nuclear extracts from rhinovirus 16-infected A549 cells that were
absent in nuclear extracts from uninfected cells. Time course
experiments demonstrated that induction of these complexes was maximal
30 min after infection and decreased with longer incubations up to
2 h (Fig. 9A).
Competition experiments were then carried out to confirm the
specificity of the binding. The addition of excess unlabeled specific
(199/170) oligonucleotide blocked binding of both of the protein
complexes (Fig. 9B, lanes 1 and
2), confirming the specificity of the binding.
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Fig. 9.
Rhinovirus infection of A549 respiratory
epithelial cells induces binding of nuclear transcription factors to
the 187/178 NF-B site in the ICAM-1
promoter within 30 min of infection. A, the time course
of induction by rhinovirus of nuclear transcription factors binding to the 199/170 (containing a C/EBP and an NF-B
binding site, Table II) portion of the ICAM-1 promoter was assessed by
EMSA. Nuclear extracts were prepared from uninfected (time 0) and
rhinovirus 16-infected A549 cells at various time points (30, 60, 90, and 120 min) after infection and incubated with radiolabeled
199/170 ICAM-1 (Table II) probe. Resolution of binding complexes
was accomplished on 5% polyacrylamide gels. A representative
radiograph of one of at least three separate experiments is shown. Two
retarded complexes binding to the 199/170 portion of the ICAM-1
promoter were induced in nuclei from rhinovirus-infected cells, with
peak induction of binding activity observed within 30 min of rhinovirus
infection. Induction of binding activity by rhinovirus reduced
gradually from 30 min but was still present up to 120 min after
infection. B, the specificity of the binding activity
induced by rhinovirus infection of A549 cells was examined by EMSA with
competition studies. Nuclear extracts from A549 cells uninfected
(lane 5) or rhinovirus 16 infected for 30 min
(lanes 1-4) were incubated with radiolabeled
199/170 ICAM-1 probe in the absence (lane 1)
or in the presence of excess unlabeled specific 199/170 probe (SC,
lane 2) and excess unlabeled probes containing
consensus NF-B and AP-1 binding sequences (lanes
3 and 4, respectively). Resolution of binding
complexes was accomplished on 5% polyacrylamide gels. A representative
radiograph of one of at least three separate experiments is shown.
Specificity of the binding activity induced by rhinovirus infection of
A549 cells to the 199/170 portion of the ICAM-1 promoter was
confirmed by complete inhibition of the induction of binding activity
with excess unlabeled specific probe (lanes 1 and
2). Involvement of the 187/178 NF-B site in the
ICAM-1 promoter in this binding activity was suggested by complete
inhibition of induced binding activity by excess unlabeled consensus
NF-B probe (lane 3) and the lack of
competition with excess unlabeled irrelevant probe (consensus AP-1;
lane 4). C, to determine whether the
187/178 NF-B or the 199/196 C/EBP binding sites were
responsible for the rhinovirus-induced binding activity, further
competition experiments were carried out with probes containing
mutations of both these sites. Nuclear extracts from A549 cells
uninfected (lane 2) or rhinovirus 16 infected for
30 min (lanes 3-5) were incubated with
radiolabeled 199/170 ICAM-1 probe in the absence (lane
3) or in the presence of excess unlabeled competitor with a
mutated (M1) C/EBP binding site 199/196
(lane 4) or unlabeled competitor with a mutated
(M2) NF-B binding site 187/178 (lane
5). Resolution of binding complexes was accomplished on 5%
polyacrylamide gels. A representative radiograph of one of at least
three separate experiments is shown. That rhinovirus infection of A549
cells induced binding of nuclear transcription factors to the
187/178 NF-B site in the ICAM-1 promoter was confirmed by the
fact that binding activity was completely abolished by competition with
excess probe containing an intact 187/178 NF-B site but a
mutated 199/196 C/EBP site (M1, lane
4) but was not affected by competition with excess probe
containing an intact 199/196 C/EBP site but a mutated 187/178
NF-B site (M2, lane 5).
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Further competition experiments were carried out to identify the
transcription factors binding to the probe. The addition of unlabeled
consensus NF-B probe completely blocked the binding of complexes to
the specific probe, while the addition of an unlabeled consensus AP-1
probe had no effect (Fig. 9B, lanes 3 and 4), suggesting that both binding complexes were formed
of proteins binding to the ICAM-1 187/178 NF-B binding site.
In order to confirm this, competition experiments with 190/170
probes containing mutated NF-B and C/EBP sites were then carried
out. Binding of these complexes was not affected by competition with
the probe containing an intact C/EBP site but a mutated ICAM-1 NF-B
site (M2), but binding was completely abrogated by
competition with the probe containing an intact NF-B site but a
mutated C/EBP site (M1) (Fig. 9C,
lanes 3-5). These results confirmed that
rhinovirus infection of A549 cells induces transcription factors
binding to the 187/178 NF-B cis element but not the 199/196
C/EBP element in the ICAM-1 promoter.
To determine which members of the NF-B/Rel protein family were
responsible for the formation of the two inducible nuclear complexes,
specific antisera directed against members of the NF-B/Rel family
(p50, p52, p65, c-Rel, and Rel-B) were studied. Antiserum specific for
p65 clearly supershifted DNA-protein complexes, while the antiserum
directed against p50 and c-Rel significantly diminished the formation
of the inducible complexes (Fig. 10).
In contrast, antibodies to p52, Rel-B and preimmune serum had no
significant effect on complex formation. These studies demonstrated
that the most important members of the NF-B/Rel family mediating
rhinovirus-induced NF-B element binding were p65, c-Rel, and p50,
with p65 being the major component of the homo- or heterodimers
formed.
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Fig. 10.
Identification of p65 as the major component
of Rel/NF-B proteins binding to 199/170
ICAM-1 probe after rhinovirus infection of A549 respiratory epithelial
cells. Nuclear lysates prepared from 30-min uninfected
(lane 1) or rhinovirus 16-infected
(lanes 2-8) A549 cells were incubated with
radiolabeled 199/170 ICAM-1 probe in the absence (lanes
1 and 2) or in the presence of antisera against
NF-B family proteins p65 (lane 3), p50
(lane 4), p52 (lane 5),
Rel-B (lane 6), c-Rel (lane
7), or preimmune serum (lane 8).
Rhinovirus infection of A549 respiratory epithelial cells for 30 min
markedly induced binding of two complexes (arrows) in
lanes 2 (no antiserum), 5, and
6 (antisera to p52 and Rel-B, respectively) and in
lane 8 (preimmune serum). In lane
3 (antiserum to p65), there was a clear supershift of both
binding complexes, associated with a marked reduction in binding at
both original sites (arrows), indicating that p65 was a
major component of both binding complexes. In lane
4 (antiserum to p50), there was a reduction in intensity of
binding at the lower original site, while in lane
7 (antiserum to c-Rel), there was a reduction in binding at
the upper original site, indicating that the binding at the upper and
lower sites was also contributed to by c-Rel and p50,
respectively.
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227 to 200 probe Containing an Sp1 Binding Site
(206/201)--
The EMSA resulted in the retardation of two
complexes, but no induction was observed in nuclear extracts from
rhinovirus-infected cells at any time points up to 2 h (data not
shown), indicating that proteins binding to this DNA segment containing
an Sp1 binding site are not induced by rhinovirus infection of A549 cells.
294 to 266 Probe Containing an AP-1 Binding Site
(284/278)--
A single protein-DNA complex was clearly induced in
nuclear extracts from rhinovirus-infected A549 epithelial cells
compared with noninfected cells, induction being maximal at 30 min and fading thereafter (data not shown). Competition experiments with specific and consensus AP-1 competitors completely abrogated the signal, while an irrelevant (Sp1) competitor did not, confirming the
AP-1 specificity of the signal (data not shown). These data suggest
that proteins binding to the AP-1 motif at 284/278 in the ICAM-1
promoter are also induced in the nuclei of A549 cells by rhinovirus infection.
74 to 43 Probe Containing an NF-B Binding Motif (62/53)
Overlapping with an Sp1 Binding Motif (59/54)--
Two retarded
complexes were induced in nuclear extracts from rhinovirus-infected
A549 cells, with a maximal induction again observed at 30 min (data not
shown). This binding activity could be competed away by excess
unlabeled specific competitor identical oligonucleotide or by excess
unlabeled oligonucleotide containing an NF-B consensus binding motif
(data not shown). The binding was unaffected by a excess unlabeled
double-stranded oligonucleotides containing an Sp1 consensus binding
motif, suggesting that rhinovirus infection of A549 cells induces
nuclear proteins binding to the 62/53 NF-B site of the ICAM-1
promoter but not the 59/54 Sp1 site.
Having observed induction of proteins capable of binding to different
transcription factor binding sites (both NF-B and AP-1) within the
ICAM-1 promoter, we then carried out reporter gene assays to determine
which of the potential candidate transcription factor binding sites
were functional in rhinovirus induction of ICAM-1 promoter activity.
Identification of the 187/178 NF-B Site as the Rhinovirus
Response Region in the ICAM-1 Promoter--
CAT constructs containing
serial deletions of the ICAM-1 promoter were studied to identify the
ICAM-1 promoter regions involved in rhinovirus induction of ICAM-1
promoter activity. As seen in Fig. 11,
CAT constructs under the control of the proximal 1160 and 277 bp of
the ICAM-1 promoter were strongly and similarly induced by rhinovirus
16 infection of A549 cells. However, further deletions of the ICAM-1
promoter to 182 bp or shorter completely abolished the capacity of
rhinovirus infection to induce ICAM-1 promoter activity. These studies
indicated the presence of DNA sequences necessary for rhinovirus
induction of ICAM-1 promoter activity between positions 277 and 182
relative to the transcription initiation site.
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Fig. 11.
Effect of rhinovirus infection of A549
respiratory epithelial cells on ICAM-1 promoter activity and
localization of promoter regions essential for induction of ICAM-1
promoter activity by rhinovirus. A549 cells were transiently
transfected with full-length (1160) and serially deleted (277,
182, 135, and 88) ICAM-1 promoter-CAT constructs and incubated
with medium alone (C) or rhinovirus 16 at an MOI of 1 (RV) for 24 h. Epithelial cells were harvested, and CAT
activity in protein-equivalent cell lysates was assessed as described
under "Materials and Methods." ICAM-1 promoter activation is
expressed as -fold induction of CAT activity in infected over control
cells. Data are mean ± S.E. of at least five separate
experiments. Rhinovirus infection of A549 respiratory epithelial cells
induced marked and similar increases in ICAM-1 promoter activity when
CAT constructs containing full-length ICAM-1 promoter or the 277
deletion of the ICAM-1 promoter were studied. Deletions to 182 and
shorter displayed no response to rhinovirus infection, indicating that
rhinovirus response elements in the ICAM-1 promoter are located between
182 and 277 base pairs from the transcription initiation
site.
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The 187/ |
TOP
ABSTRACT
INTRODUCTION