From the University Medicine, University of Southampton, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom
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ABSTRACT |
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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- 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.
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 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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C.
Oligonucleotides used in PCR analyses
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).
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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 |
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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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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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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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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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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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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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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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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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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.
|
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.
|
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.
|
The 187/
178 NF-
B binding motif, which is already known to play a
role in ICAM-1 induction by cytokines (31, 32), is located within this
region of the ICAM-1 promoter, and its sequence is truncated by the
182 deletion. Furthermore, the EMSAs clearly demonstrated rhinovirus
induction of nuclear proteins binding to this site. Therefore, for
further investigations, constructs were used that specifically tested
this site.
First, we examined whether DNA fragments containing either the complete
binding motif or the 182 deletion of the motif could confer
responsiveness to rhinovirus 16 in a heterologous promoter, the herpes
simplex minimal thymidine kinase promoter contained in the plasmid
pBRAMScat2 (31). Constructs containing the complete NF-
B
187/
178
site (pBRAMScat2
199/
170 ICAM-1), the truncated site (pBRAMScat2
199/
182 ICAM-1), and the minimal promoter alone with no ICAM-1
promoter sequence (pBRAMScat2) were transfected in A549 cells. Fig.
12 shows that only when the entire
ICAM-1
187/
178 NF-
B site is present (pBRAMScat2
199/
170
ICAM-1), is the heterologous thymidine kinase promoter responsive to
rhinovirus, confirming that this site is required intact for rhinovirus
induction of ICAM-1 promoter activity and that it is also sufficient in
the presence of a heterologous minimal promoter.
|
Finally, to investigate whether the 187/
178 NF-
B site in the
ICAM-1 promoter is essential for rhinovirus induction of ICAM-1 promoter activity to occur, constructs containing either the longest ICAM-1 promoter sequence (Fig. 13,
1160 ICAM-1) or the same construct with mutations from
TGGAAATTCC to TctAgATTag at the
187/
178 NF-
B site (
1160
mICAM-1) were used to transfect A549 cells. As shown in Fig. 13,
mutation of the
187/
178 NF-
B site completely abrogated
rhinovirus induction of ICAM-1 promoter activity, confirming that this
NF-
B binding site is necessary for rhinovirus induction of ICAM-1
gene transcription.
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DISCUSSION |
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In these studies, we have investigated mechanisms involved in rhinovirus-induced asthma exacerbations by studying the effect of rhinovirus infection on airway epithelial cell ICAM-1 expression. These studies were performed, since ICAM-1 is the receptor for 90% of rhinoviruses and is an adhesion protein that has a central role in inflammatory cell recruitment to the lower airway following rhinovirus infection. ICAM-1 is therefore likely to play a very important role in the mechanisms of virus-induced asthma exacerbations.
We have demonstrated that rhinovirus infection of both primary
bronchial epithelial cells and the type II respiratory epithelial cell
line A549 markedly increases cell surface expression of ICAM-1. We then
investigated the mechanisms of rhinovirus regulation of ICAM-1
expression in A549 cells and observed induction of ICAM-1 promoter
activity and increased ICAM-1 mRNA transcription. The rhinovirus
induction of ICAM-1 promoter activity was found to involve
up-regulation of NF-B proteins binding to the
187/
178 NF-
B
binding site on the ICAM-1 promoter, and this site was required intact
for rhinovirus up-regulation to occur. The principal component of the
rhinovirus-induced NF-
B-binding proteins were p65 subunits. These
studies elucidate mechanisms probably involved in rhinovirus induction
of asthma exacerbations and identify ICAM-1 and NF-
B p65 as
potential new targets for development of therapeutic intervention strategies for virus-induced asthma.
Our initial studies demonstrated that rhinovirus infection of A549 respiratory epithelial cells increased the cell surface expression of ICAM-1 protein in a dose-response manner, with peak induction occurring at an MOI of 1 (Fig. 1). The observed increase in ICAM-1 expression peaked at 8 h after virus inoculation but remained elevated above noninfected cells for up to 72 h after inoculation (Fig. 2).
We then investigated the group and serotype specificity of the induction of ICAM-1 and demonstrated that both major group and minor group rhinoviruses and at least three different serotypes are equally able to up-regulate ICAM-1 surface protein expression (Fig. 4). Since major and minor group viruses bind to different cellular receptors (26, 27, 35), the observed induction of ICAM-1 by rhinoviruses is clearly not receptor- or serotype-restricted. These observations are in keeping with previous observations relating to rhinovirus induction of IL-8 (14, 16, 17) and IL-6 (15, 17) and suggest that the mechanisms involved are likely to have broad applicability across all rhinovirus serotypes. Furthermore, since rhinoviruses are responsible for two-thirds of asthma exacerbations in which a virus is identified (2-4), the mechanisms involved are likely to be pertinent to the majority of asthma exacerbations.
The observation that rhinovirus infection of respiratory epithelial
cells up-regulated ICAM-1 expression is of great interest, since this
molecule is not only important in inflammatory cell recruitment and
activation in asthma (23-25) but is also the receptor for the major
group (90%) of rhinoviruses (26, 27). Induction of increased
expression of its own cell surface receptor is an unusual property for
viruses, since previous observations demonstrate that virus infection
down-regulates expression of virus cell surface receptors. For example,
measles virus and human immunodeficiency virus both induce
down-regulation of their receptors CD46 and CD4, respectively, a
process thought to prevent superinfection (40, 41). Our findings that
rhinoviruses induce increased expression of their own receptor suggest
that the converse occurs and that rhinovirus infection may render cells
more, rather than less, susceptible to infection by other major group
virus particles. A recent report goes some way toward confirming this
hypothesis, in that rhinovirus infection of primary cultures of human
tracheal epithelium was found to increase ICAM-1 mRNA expression
and similar magnitude increases in ICAM-1 mRNA expression induced
by IL-1 were found to increase susceptibility to rhinovirus
infection (42). We were therefore interested in investigating whether our finding of rhinovirus-induced increased ICAM-1 expression in A549
cells could also be observed in primary bronchial epithelial cells. We
observed a much greater induction of ICAM-1 protein (12- versus 3-fold) in the primary cells (Figs. 1-4), suggesting that any observations made in A549 cells might actually be more rather
than less marked in primary cells. Given that rhinovirus infection of
respiratory epithelial cells is low grade and noncytopathic (16),
induction of the viral receptor is likely to render the cells more
susceptible to infection and, therefore, further ICAM-1 up-regulation,
creating a "vicious cycle" of rhinovirus infection and ICAM-1
up-regulation. The effect of rhinovirus infection on the expression of
the minor group rhinovirus receptor (35) has not been studied, but
similar mechanisms could in theory also operate for minor group
rhinoviruses. This process, combined with increased ICAM-1 expression
in asthma (20-22) is likely to play an important role in increasing
the susceptibility of asthmatic subjects to lower airway rhinovirus infections.
We have previously reported rhinovirus induction of IL-8 protein and mRNA from rhinovirus-infected A549 cells and used sICAM-coated and UV-inactivated virus to investigate the relative contributions of virus-receptor binding and virus replication to the observed up-regulation of IL-8 (16). We reported that UV inactivation reduced by about two-thirds, while precoating the virus with soluble receptor completely abrogated, the induction of IL-8. These data suggest that part of the observed up-regulation of IL-8 was related purely to virus-receptor binding, while the remainder required viral replication (16). In the present studies, we observed the same findings relating to sICAM and UV inactivation to be true for rhinovirus induction of ICAM-1 and have extended them to demonstrate that filtering virus particles but not substances with a molecular mass of <30 kDa (most cytokines) from the inoculum completely abrogated the observed ICAM-1 induction (Fig. 4). We also confirmed the receptor specificity of the sICAM inactivation by demonstrating that precoating a minor group (rhinovirus 2) virus with sICAM had no effect on ICAM-1 up-regulation (Fig. 4). These additional data add further weight to our previous hypothesis that part of the signal to up-regulate ICAM-1 or IL-8 protein synthesis occurs consequent to virus-receptor binding, but the major part occurs through processes associated with viral replication.
The ability of rhinovirus infection to up-regulate epithelial cell surface expression of ICAM-1, an important molecule in asthma pathogenesis (23), may have particular importance in the mechanisms of virus-induced asthma exacerbations independent of the effects on rhinovirus replication. We have previously demonstrated that rhinovirus colds induce bronchial mucosal CD3+, CD4+, and CD8+ lymphocyte and eosinophil infiltration, with a more persistent intraepithelial eosinophilia in asthmatic subjects (7). Epithelial expression of ICAM-1 is likely to play an important function in retaining both types of inflammatory leukocyte in the epithelium by binding to its ligands CD18/11a and CD18/11b on lymphocytes and granulocytes, respectively. In addition, binding of ICAM-1 to its integrin ligands on leukocytes may activate these cells and lead to secretion of proinflammatory cytokines and mediators (43-46). Induction of ICAM-1 expression on respiratory epithelial cells is therefore likely to be an important mechanism regulating the bronchial mucosal CD3+, CD4+, and CD8+ lymphocyte and eosinophil infiltration observed in rhinovirus infections. Given the important regulatory role of lymphocytes in promoting airway inflammation in asthma, the induction by rhinoviruses of ICAM-1 is a mechanism that represents an attractive target for development of therapeutic interventions aimed at reducing inflammatory cell recruitment and activation in virus-induced asthma exacerbations.
The observations on the time course and lack of receptor or serotype restriction of ICAM-1 induction by rhinoviruses are in keeping with previous reports of rhinovirus induction of proinflammatory cytokine expression in respiratory epithelial cells (14-17). These similarities suggest that there may be common mechanisms involved in the induction of several proinflammatory proteins by rhinoviruses and that further investigation of the cellular/molecular mechanisms involved might lead to the identification of common pathways suitable for targeting of further future therapeutic intervention strategies. Therefore, having demonstrated that rhinovirus infection of respiratory epithelial cells increased ICAM-1 surface protein expression, we wished to investigate the effects of rhinovirus infection on A549 cell ICAM-1 mRNA expression to elucidate the cellular mechanisms involved in more detail. We observed rhinovirus induction of ICAM-1 mRNA occurring within 1 h of virus inoculation and peaking at 8 h (Fig. 6, Table III). Rhinovirus induction of ICAM-1 mRNA was observed up to 24 h post-virus inoculation (no studies were carried out beyond this time point). As we had observed with ICAM-1 protein expression, UV inactivation partially abrogated the rhinovirus-induced ICAM-1 mRNA expression while inactivating the virus by filtration or by precoating with soluble receptor completely abrogated the signal (Fig. 7, Table IV). These studies confirmed that, as with ICAM-1 protein expression, rhinovirus induction of ICAM-1 mRNA was also consequent partly upon virus-receptor binding and was partly related to viral replication.
Having observed rhinovirus-induction of both ICAM-1 protein and
mRNA expression, we hypothesized that rhinovirus infection of A549
cells increased ICAM-1 expression by up-regulating ICAM-1 gene
transcription. To investigate this possibility, we analyzed in
vitro transcription of ICAM-1 mRNA in rhinovirus-infected and noninfected cells. As seen in Fig. 8 and Table V, we observed clear
induction of ICAM-1 gene transcription by rhinovirus infection and
inhibition of this induction by an inhibitor of RNA polymerase II,
-amanitin. These data confirmed that rhinovirus infection of A549
respiratory epithelial cells rapidly increased de novo transcription of ICAM-1 mRNA.
Next, we wished to determine the molecular mechanisms involved in
rhinovirus induction of ICAM-1 mRNA transcription, since these
mechanisms might identify a target for development of new therapeutic
intervention strategies. We focused on transcription factor-mediated
activation of the ICAM-1 promoter, since previous observations
indicated that both IL-6 induction by rhinoviruses (15) and IL-8
induction by respiratory syncytial virus (47) were dependent on
proteins binding to NF-B sites in the relevant promoters, and our
own observations had indicated that the presence of both an NF-
B
site and an AP-1 site was required for induction of the IL-8 promoter
by rhinovirus infection (48).
The ICAM-1 promoter contains several potential transcription factor
binding sites, several of which have been implicated in the induction
of ICAM-1 gene transcription in response to various proinflammatory
stimuli, such as PMA and cytokines (31, 32). In the present study, we
observed that rhinovirus infection of A549 epithelial cells induced
proteins binding to the both the 187/
178 and the
62/
53 ICAM-1
NF-
B sites (Fig. 9 and data not shown) and the
284/
278 AP-1 site
(data not shown) but neither the
206/
201 nor
59/
54 Sp1 site. We
therefore performed reporter gene assays to determine which of these
sites was functional in rhinovirus induction of ICAM-1 promoter
activity. We observed that sequential deletion of the ICAM-1 promoter
up to
277 base pairs from the transcription initiation site had no
effect on rhinovirus induction of ICAM-1 promoter activity (Fig. 11).
These data suggest that despite the fact that proteins binding to this site are induced by rhinovirus infection of A549 cells, the
284/
278 AP-1 site is not required for rhinovirus induction of ICAM-1 promoter activity to occur. In contrast, deletion of the promoter to
182 bases
resulted in complete loss of rhinovirus inducibility, suggesting that
elements contained within the
182 to
277 region were necessary for,
and that the
62/
53 NF-
B site alone was insufficient for, rhinovirus-induced up-regulation of ICAM-1 promoter activity to occur
(Fig. 11).
The 182 deletion interrupts the
187/
178 NF-
B site. Given the
previously observed functionality of this site in ICAM-1 induction by
cytokines (32), we hypothesized that this site might also be important
in rhinovirus induction of ICAM-1 promoter activity. Mutational
analysis was therefore carried out, with reporter gene assays being
performed with full-length ICAM-1 promoter and with full-length ICAM-1
promoter with the
187/
178 NF-
B site mutated in four nucleotide
positions. Despite the presence of the full-length promoter, mutation
of this
187/
178 NF-
B site completely abrogated rhinovirus
induction of ICAM-1 promoter activity (Fig. 13), confirming that this
site was required intact for rhinovirus induction of ICAM-1 promoter
activity to occur. The importance of this
187/
178 NF-
B site in
rhinovirus induction of promoter activity was then investigated using
plasmids containing the reporter gene linked to the thymidine kinase
minimal promoter alone and with the truncated and intact versions of
the
187/
178 NF-
B site (Fig. 12). These data confirmed that this
site was required intact for rhinovirus induction of a heterologous
promoter to occur and that it was sufficient in the presence of a basic
minimal promoter.
Previous studies have demonstrated that members of the NF-B family
of transcription factors are important in induction of proinflammatory
cytokines by both rhinovirus and respiratory syncytial virus (15, 47,
48). These data suggest that NF-
B may play a very important role in
the induction of proinflammatory cytokines by virus infections in
general, and our data reported herein extend these observations to
include proinflammatory adhesion molecules. The NF-
B/Rel family of
transcription factors contains several members, so far including p50,
p52, p65, c-Rel, and Rel-B, which are capable of forming homo- or
heterodimers. We performed supershift experiments to investigate which
members of the NF-
B/Rel family were induced by rhinovirus infection
and demonstrated that the major component of rhinovirus-induced
proteins binding to the
187/
178 NF-
B site in the ICAM-1 promoter
were p65 proteins, with smaller amounts of p50 and c-Rel (Fig. 10).
These data support previous observations that the major
rhinovirus-induced proteins binding to the IL-6 promoter in A549 cells
are also p65 homodimers or heterodimers (15).
In addition to the early important study in primates by Wegner et al. (23), two further recent studies have reported important roles for ICAM-1 in promoting inflammation in asthma by demonstrating its important role in promoting lymphocyte and eosinophil infiltration in murine models (24, 25). Given the marked bronchial lymphocyte and eosinophil infiltration observed in virus-induced asthma (7), its role as the rhinovirus receptor, and our demonstration that rhinovirus infection induces increased ICAM-1 expression in respiratory epithelial cells, we believe that ICAM-1 is likely to play a critical role in promoting lower airway inflammation in virus-induced asthma. This hypothesis is supported by the fact that we2 and others (49) have observed increased sICAM levels in nasal secretions during rhinovirus infections. Recent support for the role of ICAM-1 up-regulation in rhinovirus-induced asthma also comes from the demonstration that experimental rhinovirus infection of asthmatic subjects up-regulates ICAM-1 expression on bronchial epithelial cells in vivo (50). These data make epithelial ICAM-1 a prime target for therapeutic intervention strategies in virus-induced asthma exacerbations. The fact that anti-ICAM-1 monoclonal antibodies were able to reduce allergen-induced airway hyperreactivity and eosinophil influx in a primate model of asthma (23) also strongly supports the potential of ICAM-1-targeted intervention to ameliorate virus-induced asthma exacerbations.
We have also investigated the mechanisms of rhinovirus induction of
ICAM-1 and have observed an important role for NF-B-mediated transcriptional up-regulation, the major component of which is contributed by p65. Given that transcriptional up-regulation via NF-
B is also necessary for induction of IL-6 (15) and IL-8 (48) by
rhinoviruses and that in the case of IL-6 p65 is again the major
component, this molecule also represents an important potential target
for future therapeutic intervention in virus-induced asthma exacerbations.
In conclusion, we have demonstrated that rhinovirus infection of
respiratory epithelial cells increases surface ICAM-1 expression via
NF-B p65-mediated transcriptional up-regulation. We believe that
these two molecules (ICAM-1 and NF-
B p65) represent new targets for
potential therapeutic intervention in virus-induced asthma exacerbations.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Klaus Degitz and Stephen Wright Caughman for generous donations of the reporter plasmids.
![]() |
FOOTNOTES |
---|
* This work was supported by National Asthma Campaign Grant 332 and by the University of Ferrara, Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University Medicine
(810), Southampton General Hospital, Southampton SO16 6YD, United
Kingdom. Tel.: 01703 794607; Fax: 01703 701771; E-mail: slj1{at}soton.ac.uk.
2 A. Papi and S. L. Johnston, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; ICAM-1, intercellular adhesion molecule-1; sICAM-1, soluble ICAM-1; TCID50, tissue culture infective dose 50%; APRT, adenine phosphoribosyltransferase; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; MOI, multiplicity of infection; DTT, dithiothreitol; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; bp, base pair(s); C/EBP, CCAAT/enhancer element-binding protein.
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REFERENCES |
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