Role of p38 Mitogen-Activated Protein
Kinase in Rhinovirus-Induced Cytokine
Production by Bronchial Epithelial Cells
This information is current as
of June 17, 2017.
Sandra D. Griego, Cynthia B. Weston, Jerry L. Adams, Ruth
Tal-Singer and Susan B. Dillon
J Immunol 2000; 165:5211-5220; ;
doi: 10.4049/jimmunol.165.9.5211
http://www.jimmunol.org/content/165/9/5211
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References
Role of p38 Mitogen-Activated Protein Kinase in
Rhinovirus-Induced Cytokine Production by Bronchial
Epithelial Cells1
Sandra D. Griego,2* Cynthia B. Weston,* Jerry L. Adams,† Ruth Tal-Singer,* and
Susan B. Dillon*
H
uman rhinovirus (HRV),3 the most frequent cause of the
common cold, is increasingly associated with more serious sequelae including exacerbations of asthma,
chronic bronchitis, chronic obstructive pulmonary disease, otitis
media, and sinusitis (1–3). Recent studies in adults and adolescents
using PCR to assist in viral detection have shown that up to 50 –
80% of asthma exacerbations are associated with upper respiratory
tract virus infection, and that rhinovirus is the most common isolated virus (4, 5). Respiratory epithelium represents the primary
site of replication for rhinovirus. However, only a small fraction of
upper respiratory epithelial cells are demonstrably infected, and
with minimal epithelial cell damage (6). It has been hypothesized
that the symptoms associated with HRV infection are due primar-
Departments of *Molecular Virology and Host Defense and †Medicinal Chemistry,
SmithKline Beecham Pharmaceuticals, Collegeville, PA 19426
Received for publication February 3, 2000. Accepted for publication August 10, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was presented in part by Sandra D. Griego, Cynthia Weston, Jerry L.
Adams, and Susan B. Dillon at the American Thoracic Society’s 96th International Conference, May 5–10, 2000, in Toronto, Ontario, Canada (Abstract
127622-A).
2
Address correspondence and reprint requests to Dr. Sandra D. Griego, SmithKline
Beecham Pharmaceuticals, 1250 South Collegeville Road, P.O. Box 5089, UP1450,
Collegeville, PA 19426-0989. E-mail address: [email protected]
3
Abbreviations used in this paper: HRV, human rhinovirus; MAP, mitogen-activated
protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase;
BEGM, bronchial epithelial growth media; HRV-39, rhinovirus serotype 39; TCID50,
tissue culture-infective dose of virus required to infect 50% of monolayers; RSV,
respiratory syncytial virus; GRO␣, growth-related oncogene-␣; ENA-78, epithelial
neutrophil-activating protein-78; RT, reverse transcriptase; XTT, sodium-3⬘-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate;
SB 239063, trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin4-yl)imidazole; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)
imidazole; SKF 106978, 2-(4-methylsulfinyl)-3-(4-(2-methylpyridyl))-6,7-dihydro(5H)
pyrrolo(1,2-a)imidazole.
Copyright © 2000 by The American Association of Immunologists
ily to the host response to infection rather than to an acute cytotoxic effect. In normal subjects, rhinovirus infection elicits local
infiltration of neutrophils and intranasal production of many cytokines/chemokines including IL-8, IL-6, TNF-␣, IFN-␣, RANTES,
GM-CSF, and IL-1, which peak during active virus replication (1,
7). Rhinoviruses, as well as other respiratory pathogens, have also
been shown to stimulate production of IL-6, IL-8, and GM-CSF in
cultures of primary respiratory epithelial cells or human epithelial
cell lines such as BEAS-2B (8 –11) and to increase ICAM-1 expression (12, 13). Understanding the cell signaling pathways and
factors leading to epithelial cell gene expression in response to
rhinovirus infection may provide new therapeutic strategies for the
treatment of inflammation associated with respiratory virus
infections.
The p38 kinase, a serine-threonine kinase, is a member of the
mitogen-activated protein (MAP) kinase superfamily, which also
includes extracellular signal-regulated kinase (ERK) and stressactivated protein kinase/c-Jun N-terminal kinase (JNK). The MAP
kinases are important mediators of signal transduction, which regulate gene expression through a cascade of protein phosphorylation events, thereby inducing a variety of cellular responses such as
apoptosis, proliferation, and cytokine biosynthesis. The p38 kinase
is activated in a variety of cell types in response to hyperosmotic
shock, growth factors, LPS, and proinflammatory cytokines and
has been implicated in the downstream activation of multiple transcription factors such as activating transcription factor 2, Elk-1,
C/EBP homologous protein, and cAMP response element binding
protein (reviewed in Refs. 14 and 15). Inhibition of p38 kinase
activity in various cell types with specific pharmacologic agents
(pyridinyl imidazoles) has been shown to block production of inflammatory mediators such as IL-1, TNF, IL-6, IL-8, and GM-CSF
through regulation of transcriptional and/or translational events
(14). Several recent studies have suggested a role for p38 kinase in
0022-1767/00/$02.00
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The stress-activated protein kinase p38 plays a central role in the regulation of cytokine biosynthesis by various cell types in
response to a wide range of stimuli. Because the local inflammatory response and the infiltration of neutrophils is thought to
contribute to the symptoms and sequelae of rhinovirus infection, we investigated the role of p38 kinase in cytokine and chemokine
elaboration in airway epithelial cells infected with human rhinovirus. Rhinovirus-39 infection of BEAS-2B cells resulted in synthesis of cytokines (IL-1, IL-6, G-CSF, and GM-CSF) and CXC chemokines (IL-8, epithelial neutrophil-activating protein-78, and
growth-related oncogene-␣), evident 24 –72 h postinfection. Rhinovirus infection induced a time- and dose-dependent increase in
tyrosine phosphorylation of p38 kinase, which peaked 30 min postinfection and remained elevated for 1 h. Treatment of infected
cells with SB 239063, a potent pyridinyl imidazole inhibitor of p38 kinase, resulted in up to 100% inhibition of mediator production
and partially reduced levels of IL-8 mRNA as determined by quantitative RT-PCR. Treatment with SB 239063 had no effect on
virus replication and was not cytotoxic at concentrations < 70 ␮M. These studies provide the first evidence that early activation
of p38 kinase by rhinovirus infection is a key event in regulation of virus-induced cytokine transcription, and may provide a new
target for inhibition of symptoms and airway inflammation associated with rhinovirus infection. The Journal of Immunology,
2000, 165: 5211–5220.
5212
airway inflammation through induction of IL-8 expression in bronchial epithelial cells (16, 17).
In this study we examined the role of the p38 kinase signal
transduction pathway on cytokine and chemokine expression in a
human bronchial epithelial cell line, BEAS-2B, in response to infection with rhinovirus. These studies have demonstrated that multiple CXC-chemokines (epithelial neutrophil-activating protein-78
(ENA-78), growth-related oncogene-␣ (GRO␣), and IL-8) and cytokines (G-CSF and GM-CSF) that are important in neutrophil
activation and function are produced in response to rhinovirus infection. We present evidence that p38 kinase is rapidly phosphorylated in response to rhinovirus infection and that chemokine and
cytokine biosynthesis is inhibited in the presence of pharmacological inhibitors of p38 kinase activity.
p38 KINASE ACTIVATION BY RHINOVIRUS
supernatants were determined by specific ELISA using commercially
available kits according to manufacturer’s instructions (R&D Systems,
Minneapolis, MN).
Drug treatments
The p38 kinase inhibitors, SB 239063 (trans-1-(4-hydroxycyclohexyl)4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole), SB 203580
(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole),
and the control compound SKF 106978 (2-(4-methylsulfinyl)-3-(4-(2methylpyridyl))-6,7-dihydro(5H)pyrrolo(1,2-a)imidazole) were dissolved
in DMSO to provide stock solutions (10 mM), which were stored at ⫺20°C
until use. Compounds were diluted to appropriate concentrations in medium and added to cultures 30 min before infection. After removal of virus
inoculum from infected cultures, fresh compound was added with medium
and remained for the duration of the culture.
Extraction of RNA and reverse transcriptase (RT)
Materials and Methods
Cell lines
Virus and virus propagation
Rhinovirus serotype 39 (HRV-39) was purchased from American Type
Culture Collection. A stock solution of HRV-39 was prepared by infecting
monolayer cultures of HeLa cells. Cultures were grown until cytopathic
effect was ⬎80% after which cells were harvested using a cell scraper to
dislodge adherent cells, frozen at ⫺70°C, thawed once, and pelleted
through a 30% sucrose cushion by centrifugation for 2 h at 4°C at 25,000
rpm using a SW28 rotor in a Beckman Coulter (Fullerton, CA) L8 – 80
ultracentrifuge. The virus pellet was resuspended in BEGM medium and
stored in aliquots at ⫺70°C. Virus titers were determined by microtitration
using HeLa cell monolayers, as described below. In some experiments,
HRV-39 was inactivated by exposure to UV light and confirmed by microtitration on HeLa cells. Virus was irradiated in polypropylene containers
at a solution depth of 1–2 mm. Irradation was performed for 20 min at a
distance of 15 cm from a mercury germicidal lamp (Universal light source,
G36T6L/CB) emitting at 254 nm.
Virus titration
Detection of virus in BEAS-2B infected cultures or titration of virus stocks
was performed by infecting HeLa cell monolayers (104 cells/well) in 96well plates with 10-fold dilutions of virus-containing medium in MEM/
10% FCS (6 replicates/dilution). Plates were incubated for 5 days at 34°C
after which wells were scored positive or negative for cytopathic effect by
microscopic examination. Calculation of tissue culture-infective dose
(TCID50; amount of virus required to infect 50% of monolayers) was calculated according to the Spearman-Karber method.
Virus infection of BEAS-2B cells
BEAS-2B cells (2 ⫻ 105 cells/well) were cultured in collagen-coated wells
in 24-well plates for 24 h before infection with rhinovirus. HRV-39 was
added to cell cultures at 2 ⫻ 105 TCID50/well (1 TCID50/cell) unless otherwise indicated. After 1 h incubation at 34°C, virus inoculum was aspirated and replaced with fresh media and incubation of cultures at 34°C was
continued. Supernatants were collected at various times postinfection and
stored at ⫺70°C until assayed for cytokine protein concentration or virus
yield. Two to four replicate cultures were set up for each treatment or time
point, and replicates were pooled at time of harvest to provide sufficient
sample to run multiple analyses.
Quantitation of cytokines
Cell-free supernatants from control or infected BEAS-2B cultures were
harvested at various times postinfection, clarified by centrifugation (200 ⫻
g, 10 min) and stored at ⫺70°C until assayed. Levels of cytokines in
Conventional PCR amplification of cDNA
Detection of cytokine mRNA was initially conducted using standard PCR
amplification of cDNA templates generated in the RT reaction described
above. Primer sets for each cytokine (Amplimer sets) were purchased from
Clontech (Palo Alto, CA) and PCR conditions used were as recommended
by the manufacturer. Detection of rhinovirus mRNA was also initially performed using standard RT-PCR. PCR was performed using primers introduced above (18). A PCR product of the expected size (380 bp) was detected in samples generated from RV39-infected cell RNA. RNA extracted
from control uninfected cells or from cells infected with respiratory syncytial virus (RSV) did not produce any detectable PCR product at 380 bp
confirming the specificity of the OL27/OL26 primer pair for detection of
rhinovirus RNA (data not shown).
Samples with positive PCR products of the expected size were further
analyzed using quantitative real-time PCR.
Quantitative real-time PCR
Reactions were performed in triplicate containing 2⫻ Universal PCR Master Mix (Perkin-Elmer, Norwalk, CT), 1 ␮l of template cDNA, 200 nM of
primers, and 100 nM of probe in a final volume of 50 ␮l, and were analyzed in Microamp optical 96-well plates (Perkin-Elmer). Primer pairs and
probes described in Table I were designed using Primer Express software
(Perkin-Elmer). Probes were synthesized by Synthegen (Houston, TX) to
include a fluorescent reporter dye, FAM, on the 5⬘ end and labeled with a
fluorescent quencher dye, TAMRA, on the 3⬘ end to allow direct detection
of the PCR product. Reactions were amplified and quantitated using an
ABI 7700 sequence detector and manufacturer’s software (Perkin-Elmer).
Standard curve generation
Relative quantities were interpolated from a standard curve generated by
serial dilution of human genomic DNA (Clontech) or cDNA templates
generated from positive control samples in which expression of the target
mRNA had been confirmed by conventional PCR techniques, as described
above. To control for variation in the input cDNA quantity, expression of
the target cytokine mRNA was normalized to GAPDH expression, a housekeeping gene, in the same sample by dividing the quantity of PCR product
of interest by the quantity of GAPDH product. Normalized values were
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Human bronchial epithelial cells (BEAS-2B passage 36; American Type
Culture Collection, Manassas, VA) were cultured in collagen-coated culture flasks according to instructions provided using bronchial epithelial
growth medium (BEGM; Clonetics, San Diego, CA) containing 0.5 ng/ml
human recombinant epithelial growth factor, 5 ␮g/ml insulin, 0.5 ␮g/ml
hydrocortisone, 0.5 ␮g/ml epinephrine, and 10 ␮g/ml transferrin. All experiments with BEAS-2B cells were performed using cells between passages 38 and 55. HeLa cell cultures (American Type Culture Collection),
used for detection and titration of virus, were maintained in Eagle’s MEM
supplemented with 10% FCS, 2 mM L-glutamine, and 10 mM HEPES
buffer (MEM/10% FCS).
RNA was extracted from BEAS-2B cells infected with HRV-39, or control
cells, using RNA-Stat60 (Tel-Test, Friendswood, TX) according to manufacturer’s instructions. Two to four replicate cultures were pooled for each
treatment or time point tested. After drying, RNA was resuspended in
diethyl pyrocarbonate-treated water and digested with RNase-free DNase I
(GenHunter, Nashville, TN) for 30 min to degrade any trace amounts of
genomic DNA, followed by a 5-min incubation at 75°C to inactivate the
enzyme. cDNA was generated from 1 to 2 ␮g of total RNA using a
GeneAmp RNA-PCR kit (Perkin-Elmer, Foster City, CA). For experiments
measuring cytokine mRNA, the RT reaction (final volume, 20 ␮l) was
conducted for 60 min at 42°C using oligo-dT and 0.5 uM/␮l random hexamer primers. For detection of viral RNA, primers specific for either the
positive-strand RNA or negative-strand RNA (designations OL27 and
OL26, respectively) were used in the RT reaction (1 ␮g/␮l). These primers
are directed to a highly conserved 5⬘ noncoding region of the viral genome
and have been described previously (18). The resulting cDNA was aliquoted and stored frozen at ⫺70°C until use in PCR. Parallel reactions
were routinely included, in which either primers or RT enzyme were excluded to control for nonspecific priming.
The Journal of Immunology
5213
Table I. PCR primer sequences and probes for use in real-time PCR
Target Sequence
Locations bp (5⬘-3⬘)
CAA GGT CAT CCA TGA CAA CTT TG
GGC CAT CCA CAG TCT TCT GG
ACC ACA GTC CAT GCC ATC ACT GCC A
13–30
62–81
32–60
CTG GCC GTG GCT CTC TTG
CCT TGG CAA AAC TGC ACC TT
CAG CCT TCC TGA TTT CTG CAG CTC TGT GT
13–30
62–81
32–60
CCA GGA GCC CAG CTA TGA ACT
CCC CAG GGA GAA GGC AA
TCT CCA CAA GCG CCT TCG GTC C
22–42
70–86
46–67
CCC AAG AAC ATC CAA AGT CTG A
TTC GGT TTG GGC GCA G
CGT GAA GTC CCC CGG ACC CC
213–234
257–272
236–255
CCT GCT CAA GTG CTT AGA GCA A
GCT TCT CCT GGA GCG CTG
TGA GGA GAT CCA GGG GAT GGC
171–192
218–235
194–216
TGG GAG TGG CCT GGA CC
TCC CAT TCT TCT GCC ATG C
CCC TGG GCA CAC TGA CCC TGA TAC A
590–606
635–653
608–633
TGA AGA GCC GCG TGT GC
TGG CTG CAG GTT TAA GGT TAG
ACT TTG AGT CCT CCG GCC CCT GAA T
417–433
464–485
436–460
either plotted directly, or plotted as relative expression determined by dividing the quantity of PCR product in infected cells by the quantity of PCR
product in uninfected controls, using the normalized values.
Because viral RNA was transcribed into cDNA using a virus specific
primer, normalization to an internal standard (GAPDH) was not performed.
Therefore, plotted values represent quantities obtained directly from interpolation from a standard curve generated using cDNA transcribed from
1–2 ␮g RNA from HRV-39-infected HeLa cells harvested 24 h postinfection when maximum virus titer is present.
Quantitation using standard curves described above was also validated
using cDNA transcribed from PMA-stimulated human peripheral blood
mononuclear cells (to quantitate cytokine mRNA) or cDNA transcribed
from partially purified virus (to quantitate viral RNA).
XTT assay for drug cytotoxicity
Cytotoxic concentrations of the pyridinyl imidazole compounds were assessed in BEAS-2B cultures using a sodium 3⬘-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate
(XTT) cleavage assay (19). Serial 2-fold dilutions of compounds starting at
100 ␮M were added to BEAS-2B cultures in microplates and incubated for
72 h at 34°C to mimic infection protocols. Bioreduction of XTT to
formazan was determined after an additional 3-h incubation at 37°C. Absorbance was read at 450 nm using a Dynex ELISA microplate plate
reader.
Antiviral assay
Compounds were tested for antiviral activity using a standard minimum
inhibitory concentration assay (20). Twofold dilutions of compound starting at 10 ␮M were added to 50 –100 TCID50 HRV-39 in equal volumes (50
␮l each). Dilutions (100 ␮l each) were added to HeLa cell monolayers
cultured in 100 ␮l media so that final volume per well was 200 ␮l. Cultures
were incubated for 5 days at 34°C until cytopathic effect in virus control
wells (no drug) was maximal. Inhibition of cytopathic effect in the presence
of compound was assessed by microscopy.
Western blot analysis of p38 MAP kinase
For detection of p38 kinase, BEAS-2B cells (4 ⫻ 105 cells/well) in BEGM
were plated in six-well plates 1 day before infection. One hour before
infection, BEGM was removed and cultures were incubated in basal media
without growth factors and additives to reduce endogenous levels of activated p38 kinase. Rhinovirus was added at various TCID50/well, and cells
were harvested at various time points after the addition of virus. After
removal of culture supernatant, cells were lysed directly into SDS sample
buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, and
0.1% bromophenol blue). Detection of tyrosine-phosphorylated p38 kinase
by immunoblot was analyzed by a commercially available kit according to
the manufacturer’s instructions (PhosphoPlus p38 MAPK Ab Kit; New
England Biolabs, Beverly, MA). Amounts of p38 kinase phosphorylation
were quantitated by fluorimager using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
Results
Rhinovirus infection of BEAS-2B cells
To confirm active replication of HRV-39 in the BEAS-2B cells, we
measured the presence of both infectious virus recovered from
supernatant and the presence of viral RNA in infected cells at
various times postinfection with 2 ⫻ 105 TCID50 HRV-39. Infectious virus, representing unbound inoculum, was detected in virus
yield assays 1 h postinfection, but decreased over the next 6 h to ⱕ
1.5 TCID50/ml (limit of detection). Virus was again recovered
from culture supernatant 24 h postinfection and the titer recovered
at each time point progressively increased over successive 24-h
culture periods (Fig. 1). Because culture supernatant was replaced
with fresh media after each collection period, virus recovered after
the second and third day indicated continuous viral shedding
throughout the culture period. A similar pattern of virus replication
was observed using infected cell lysates, with increasing titers observed 24 –72 h postinfection (data not shown). There was no cytopathic effect observed in virus-infected cultures, and culture confluency in uninfected and virus-infected cultures was comparable
throughout the 72-h culture period (data not shown).
The kinetics of viral RNA synthesis as detected by RT-PCR
mirrored the kinetics of virus replication seen in virus yield experiments. Quantitative RT-PCR was performed using cells from
the same cultures used to measure virus titer, in which media had
been replaced at each time point. Viral RNA, representing adsorbed or internalized inoculum, was detectable 1 h postinfection
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GAPDH
Sense
Antisense
Probe
IL-8
Sense
Antisense
Probe
IL-6
Sense
Antisense
Probe
GRO␣
Sense
Antisense
Probe
G-CSF
Sense
Antisense
Probe
GM-CSF
Sense
Antisense
Probe
HRV
Sense
Antisense
Probe
Primer Sequences (5⬘-3⬘)
5214
p38 KINASE ACTIVATION BY RHINOVIRUS
but levels declined over the next 3– 6 h. By 24 h, a significant
increase in PCR products derived from both positive- and negative-strand RNA were detected, with levels gradually decreasing
over the next two 24-h time periods. However, the continued presence of detectable levels of viral RNA 48 –72 h postinfection indicated ongoing viral replication. No PCR products were generated
in RT-PCR using RNA extracted from uninfected cells or using
cDNA templates and PCR probe in the absence of PCR primers.
Viral replication was not detected by either yield experiments or
RT-PCR after exposure of the BEAS-2B cells to UV-inactivated
virus at any of the time points tested (not shown).
Cytokine protein secretion and mRNA accumulation in response
to rhinovirus infection
FIGURE 2. Cytokine production by rhinovirus-infected BEAS-2B cells 72 h after infection with HRV-39. Data represent the mean ⫾ SEM cytokine
concentration obtained from six experiments in uninfected (o) and HRV-39-infected (f) cultures.
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FIGURE 1. HRV-39 replication in BEAS-2B cells. A, Titers of infectious virus in culture supernatants harvested at various times after infection
with HRV-39 are shown (mean ⫾ SD from four experiments; o). B, Lines
represent corresponding levels of positive-strand vRNA or negative-strand
vRNA at various times postinfection, determined by RT-PCR, from a representative experiment.
The release of IL-8, IL-6, and GM-CSF by BEAS-2B cells in
response to rhinovirus infection has been reported previously by
several investigators (8, 11, 21). In addition to looking at these
factors we also sought to determine whether other chemokines and
growth factors important in neutrophil and eosinophil recruitment
or activation were elicited by rhinovirus infection. Supernatants
from cultures of BEAS-2B cells infected with HRV-39 were harvested at 72 h postinfection and assayed for cytokine protein content by ELISA. The harvest at 72 h was chosen based on preliminary studies using IL-1 or TNF stimulation of BEAS-2B cells, in
which cytokine levels continued to accumulate throughout this
time period (not shown). As noted above, cell replication (based on
culture confluency) was similar in virus-infected and uninfected
cultures during this period. In agreement with previously reported
data, significant levels of IL-8 (4,950 pg/ml), IL-6 (4,275 pg/ml),
and GM-CSF (40 pg/ml) were secreted by BEAS-2B cells in response to rhinovirus infection (Fig. 2). In addition, significant levels of G-CSF (4,307 pg/ml), and the CXC chemokines, GRO␣
The Journal of Immunology
control cells (Fig. 3; lines). Of interest, the response appeared biphasic with a second increase in mRNA observed 24 – 48 h postinfection paralleling the kinetics of vRNA replication, although the
magnitude of this second increase in cytokine mRNA varied with
different preparations of HRV-39. Cytokine mRNA levels remained elevated compared with uninfected control cells for up to
48 –72 h postinfection.
Cytokine release in response to UV inactivation of rhinovirus
To further investigate whether cytokine production was related to
virus replication, BEAS-2B cells were incubated for 72 h with
UV-inactivated virus, various TCID50 units of infectious virus, or
medium alone. As shown in Fig. 4, the cytokine response to infectious virus was dose-dependent. In addition, UV inactivation of
virus significantly diminished the ability to stimulate cytokine production at 1 TCID50/cell, although low levels of IL-8, IL-6, and
GM-CSF were produced (Fig. 4). These results suggested that replicating virus was required for induction of optimal cytokine responses and that cytokine synthesis was not significantly stimulated by other factors present in the virus preparation.
Cytokine secretion in response to RV infection is not mediated
indirectly through IL-1
Studies from other laboratories have shown that increased IL-6
cytokine production or adhesion molecule expression by virusinfected epithelial cells was mediated indirectly via induction of
FIGURE 3. Time course of induction of cytokine mRNA and protein synthesis for IL-8 (upper left), GRO␣ (upper right), G-CSF (lower left), and
GM-CSF (lower right) in response to HRV-39 infection. Bars indicate protein levels (cytokine secreted since previous time point) for uninfected BEAS-2B
cells (p) or HRV-39-infected BEAS-2B cells (o). Lines represent corresponding cytokine mRNA levels detected at each time point.
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(13,022 pg/ml) and ENA-78 (900 pg/ml), were produced by infected cells (Fig. 2). Low levels of IL-1␣ (15 pg/ml) were also
observed in some experiments whereas IL-1␤ and TNF-␣ were not
produced by infected cells (not shown). Of the mediators tested,
GRO␣ was present at the highest endogenous levels in cultures of
uninfected cells (2,440 pg/ml). Production of the CC-chemokines
eotaxin, eotaxin 2, monocyte chemoattractant protein-3, monocyte
chemoattractant protein-4, macrophage inflammatory protein-1␣, or
RANTES was not detected in culture supernatant from infected cells.
The time course of mRNA induction and protein secretion for
infected cells was analyzed for a subset of the above mediators
(IL-8, GRO␣, G-CSF, and GM-CSF). Conditioned media was collected at indicated time points and replaced with fresh media. RNA
was harvested from the same cell culture wells used to measure
protein release. A representative experiment is shown in Fig. 3.
Secretion of cytokines into the supernatant in response to infection
was initially detected by 3– 6 h postinfection; however, peak cytokine secretion occurred 48 –72 h postinfection. (Fig. 3; bars). The
significant release of GRO␣ in uninfected cells evident at the harvest at 72 h is consistent with the presence of high endogenous
levels noted in Fig. 2, suggesting that culture conditions (i.e., culture confluency) at this late time point contributed to the overall
levels of GRO␣ detected.
Rhinovirus infection also resulted in substantial increases in
mRNA expression for IL-8, GRO␣, G-CSF, and GM-CSF detectable as early as 1 h postinfection as compared with uninfected
5215
5216
IL-1␣ or IL-1␤ (12, 22). The apparent biphasic response in mRNA
induction in our studies also suggested the possibility of a secondary stimulus. To determine whether induction of low levels of
IL-1␣, IL-1␤, or TNF-␣ were responsible for the observed increases in cytokine levels in our system, the effect of neutralizing
Abs to these cytokines on IL-8 and IL-6 production in response to
HRV-39 infection was determined. The presence of neutralizing
Abs to IL-1␣, IL-1␤, and TNF-␣ had no effect on the level of
either IL-8 or IL-6 produced by rhinovirus-infected BEAS-2B
cells suggesting that rhinovirus directly stimulated production of
these cytokines (data not shown).
Inhibition of cytokine production by specific inhibitors of p38
MAP kinase
The role of the p38 kinase signal transduction pathway in rhinovirus-induced cytokine production in epithelial cells was initially
tested using the p38 kinase inhibitor, SB 203580 (14, 23). As a
control for specificity, SKF 106978, an analogue of SB 203580
devoid of p38 kinase-inhibitory activity, was also tested at the
same concentration. BEAS-2B cells that had been preincubated
with drug for 30 min were infected with HRV-39 and cytokine
concentrations in infected culture supernatants were assessed after
72 h. Compounds were present during the infection period and for
the duration of the culture. Treatment of infected cells with SB
203580 (3 ␮M) resulted in a 34 –57% decrease in release of all the
mediators tested (IL-8, GRO, G-CSF, IL-6, GM-CSF, and ENA78). SKF 106978 had minimal effect on cytokine production
(Table II), which was similar to the effect seen with the addition of
DMSO alone (0.03%; not shown).
SB 203580 has previously been shown to be ⬎1000-fold selective for p38 kinase vs a panel of protein kinases including JNK1,
ERK2, Mek-1, Cdc2, protein kinase A, protein kinase C-␤2,
TGF-␤I and II␤, MAPKAP2, LCK, and epidermal growth factor
receptor tyrosine kinase (14). However, SB 203580 demonstrated
only a 10-fold selectivity ratio against the related c-raf (IC50 of 48
nM vs 280 nM, respectively; Ref. 14). Therefore, we confirmed
our results with a second inhibitor of p38 kinase activity, SB
239063, which demonstrated potency comparable to SB 203580 in
enzyme inhibition assays (IC50 of 44 nM) but did not exhibit activity against c-raf (IC50 ⬎ 50,000 nM; Ref. 24). SB 239063 also
demonstrated greater selectivity against several of the other protein
kinases such as LCK, Cdc2, and epidermal growth factor receptor
tyrosine kinase when compared directly to SB 203580 (data not
shown). Despite comparable activity in cell-free enzyme inhibition
assays, SB 239063 treatment of HRV-infected BEAS-2B cells resulted in 76 –100% inhibition of cytokine secretion vs 34 –57%
inhibition observed with SB 203580 (3 ␮M; Table III vs Table II).
Of note, cytokine levels were higher in the series of experiments in
which SB 203580 had been tested. As shown in Table III, the dose
responses for inhibition by SB 239063 were similar for most of the
cytokines or chemokines tested (approximate IC50s of 3–30 nM).
Inhibition of GRO␣ secretion in response to HRV-39 infection
required the highest SB 239063 concentration (IC50 of 300 nM).
Collectively these results provide strong evidence that the inflammatory cytokine response to HRV-39 infection is primarily mediated through the p38 kinase signal transduction pathway.
The inhibition of cytokine production by p38 kinase inhibitors
was not due to general cell cytotoxicity as determined by standard
XTT assays (CC50 ⬎ 70 ␮M for either compound). These compounds also did not exhibit direct antiviral activity as assessed
using a standard HeLa cell antiviral assay (MIC50 ⬎ 10 ␮M for
either compound) or by measuring virus yield in the rhinovirusinfected BEAS-2B cultures directly (results not shown).
Rhinovirus causes tyrosine phosphorylation of p38 kinase
To confirm that the p38 kinase was involved directly in cell activation after infection with rhinovirus, we measured the presence of
tyrosine-phosphorylated p38 kinase by immunoblot at various
times after the addition of HRV-39 to BEAS-2B cultures. IL-1stimulated cells were used as a positive control and p38 activation
was demonstrated within 15 min posttreatment (Fig. 5A). HRV-39
infection of BEAS-2B cells also resulted in an increase in phosphorylated p38 kinase, which was both dose and time dependent.
Increases in phosphorylated p38 kinase were evident by 15 min
Table II. Inhibition of cytokine production by SB 203580
% Inhibitionb
Cytokine
Control Levels
(pg/ml)a
IL-8
4082 ⫾ 441
IL-6
3972 ⫾ 887
GM-CSF
49 ⫾ 21
ENA-78
748 ⫾ 244
GRO␣
19785 ⫾ 1000
G-CSF
5829 ⫾ 118
a
SB 203580 (3 ␮M) SKF 106978 (3 ␮M)
49 ⫾ 6
57 ⫾ 10
42 ⫾ 8
34 ⫾ 8
49 ⫾ 2
49 ⫾ 9
11 ⫾ 8
10 ⫾ 1
22 ⫾ 3
NTc
NT
NT
Cytokine concentrations in supernatants from untreated HRV-39 infected cells.
Percent inhibition is calculated as ((pg/ml in presence of drug ⫺ pg/ml in uninfected)/(pg/ml in infected cultures ⫺ pg/ml in uninfected cultures) ⫺ 1) ⫻ 100,
where 100% inhibition would result in cytokine values comparable to uninfected
cultures. Results are mean ⫾ SD of three experiments.
c
NT, Not tested.
b
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FIGURE 4. Cytokine release in response to UV-inactivated or infectious HRV-39. Data represents protein concentrations of IL-6 (top), IL-8
(middle), and GM-CSF (bottom) in supernatants from BEAS-2B cells exposed to infectious HRV-39 (1, 0.1, or 0.01 TCID50/cell), UV-inactivated
HRV-39 (1 TCID50 /cell), or media alone (representative of three
experiments).
p38 KINASE ACTIVATION BY RHINOVIRUS
The Journal of Immunology
5217
Table III. Inhibition of cytokines by treatment with SB 239063
IL-8
Treatment
pg/ml
Uninfected
HRV-39
SB 239063
3000 nM
300 nM
30 nM
3 nM
338
2178
611
1068
1272
1311
IL-6
% Inhibition
a
pg/ml
G-CSF
% Inhibition
297
1642
85
60
49
47
283
523
921
957
pg/ml
% Inhibition
687
4976
100
83
54
51
GM-CSF
1735
2459
3081
3174
pg/ml
% Inhibition
16
109
76
59
44
42
34
66
65
78
pg/ml
% Inhibition
117
470
81
46
47
33
GRO-␣
ENA-78
185
195
296
294
pg/ml
% Inhibition
1837
8859
81
78
49
50
3102
5339
6952
NTb
82
50
27
NT
a
Percent inhibition is calculated as ((pg/ml in presence of drug ⫺ pg/ml uninfected)/(pg/ml in infected cultures ⫺ pg/ml in uninfected cultures) ⫺ 1) ⫻ 100, where 100%
inhibition would result in cytokine values comparable to uninfected cultures.
b
NT, Not tested.
between all the groups indicating that virus infection caused phosphorylation of p38 kinase without de novo synthesis of protein
(Fig. 5).
Studies reported previously indicated that the pyridinyl imidazole compounds inhibit p38 kinase enzyme activity through competition with ATP (25). Consistent with this mechanism of action,
SB 239063 had no effect on the level of tyrosine phosphorylation
of the p38 kinase in BEAS-2B cells in response to HRV-39 infection suggesting that this compound affected the enzymatic activity
of the p38 kinase rather than its activation (Fig. 6).
Effect of p38 kinase inhibitors on cytokine mRNA expression
To further evaluate the effect of p38 kinase on HRV-induced cytokine production, we also wanted to determine whether inhibitors
of p38 kinase regulated cytokine production at the level of mRNA
transcription. In these studies, SB 239063 was used to treat cells
and IL-8 mRNA levels were assessed. Cytokine mRNA expression
was examined 1, 3, 6, 24, and 48 h postinfection in untreated
infected BEAS-2B cells or in infected cells treated with 3 ␮M SB
239063. HRV-induced IL-8 mRNA up-regulation was only partially inhibited in cells treated with SB 239063 (Fig. 7). These data
were reproducible in three experiments.
FIGURE 5. p38 kinase activation in response to HRV-39 infection. A,
Time course of p38 kinase activation in BEAS-2B cells incubated with 25
ng/ml HRV-39 or IL-1␤. B, Dose-dependent activation of p38 kinase in
BEAS-2B cells incubated with HRV-39 for 30 min. Western blots of tyrosine-phosphorylated vs total p38 kinase are shown in top two panels.
Bottom panels indicate densitometer volumes from control BEAS-2B cells,
HRV-39 infected cells, or cells treated with IL-1␤ (A) or densitometic
ratios of infected vs uninfected BEAS-2B cells (B; fold increase). Results
from representative experiments are shown (n ⫽ 3).
FIGURE 6. Effect of SB 239063 on tyrosine phosphorylation of p38
kinase in response to HRV-39 infection. Western blot (top panels) or densitometric ratios (bottom panel) showing amount of activated p38 kinase in
untreated HRV-39 infected cells (f) or infected cells treated with SB 239063
(o) vs untreated uninfected cells. Representative experiment (n ⫽ 2).
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postexposure to HRV-39, appeared to peak by 30 min, and remained elevated 60 min postinfection (Fig. 5A). In addition, rhinovirus-induced tyrosine phosphorylation of p38 kinase was dose
dependent (Fig. 5B). When cells were cultured in the absence of
virus, there was no increase in the amount of tyrosine phosphorylation of p38 kinase at any of the time points tested (Fig. 5A,
control). Overall levels of p38 kinase protein were comparable
5218
Discussion
Rhinovirus infection, as well as infection with other respiratory
viruses, has been shown to stimulate production of IL-6, IL-8, and
GM-CSF in nasal secretions of infected individuals and in cultures
of primary respiratory epithelial cells or in human repiratory epithelial cell lines (7, 8, 10, 11). IL-8 is thought to be primarily
responsible for the neutrophil infiltration that is evident early after
natural or experimental rhinovirus infection (7, 11). The objective
of this study was to further characterize the chemokine response to
rhinovirus infection using the BEAS-2B bronchial epithelial cell
culture system. p38 MAP kinase signal transduction pathway has
been implicated in cytokine elaboration by multiple cell types in
response to various inflammatory stimuli (reviewed in Refs. 14 and
26). Therefore, we investigated the role of this pathway in rhinovirus-induced cytokine production.
This study is the first to demonstrate that GRO␣, ENA-78, and
G-CSF, factors important in the recruitment and activation of neutrophils, are induced in the bronchial epithelial cell line, BEAS-2B,
upon active infection with rhinovirus. Consistent with previous
reports we were also able to demonstrate dose and time-dependent
increases in cytokine mRNA expression and protein secretion of
IL-8, IL-6, and GM-CSF (8, 10, 11). The level of protein induction
in BEAS-2B cells in response to rhinovirus infection was comparable for each of the mediators tested (5- to 7-fold), although
GRO␣ was produced at the highest levels endogenously by uninfected cells (Table II, Fig. 1). Cytokine mRNA accumulation initially peaked 1– 6 h postinfection. A second increase in cytokine
mRNA was observed 24 – 48 h postinfection, although the magnitude of the second signal varied between different preparations of
the virus. Cytokine mRNA levels remained elevated compared
with uninfected cells up to 72 h postinfection, consistent with the
continued release of cytokine observed 48 –72 h postinfection in
these studies.
The kinetics of cytokine secretion in our studies differed from
those described in earlier reports using BEAS-2B cells (8, 21) in
which significant cytokine release was evident before 24 h. In the
present study, low but detectable levels of cytokine were present 3
to 6 h postinfection, consistent with the early increase in mRNA
accumulation, but peak protein secretion occurred 48 –72 h postinfection. In studies by Zhu et al. (11), the kinetics and levels of IL-8
produced in response to HRV infection differed among A549,
MRC-5, and normal human bronchial epithelial cells. However,
IL-8 protein levels continued to increase with continued cell culture up to 48 –96 h postinfection. These data are consistent with the
kinetics of cytokine secretion in HRV-infected BEAS-2B cells in
the present study. HRV infection of primary bronchial epithelial
cells induced production of RANTES in culture supernatants (27).
RANTES, as well as GRO␣, was also produced in A549 cells in
response to HRV infection (28). However, we were routinely unable to detect RANTES production in response to rhinovirus. The
reason for these differences is unclear but may be attributed to
differences in virus preparations (host cell, purification, virus
strain, etc.), cell type, or culture conditions used by different
investigators.
Similar to our observations in HRV-infected BEAS-2B cells,
Fiedler et al. (29) reported a biphasic accumulation of IL-8 mRNA
in RSV-infected A549 cells. The early response was observed in
cultures inoculated with nonreplicative virus, whereas the late response was dependent on viral protein synthesis and viral replication (29). Thus, the early peak in HRV-induced transcript accumulation in the present study was possibly due to early stages of
virus-cell interactions such as binding and/or internalization. The
second peak in mRNA 24 h postinfection corresponded to HRV
replication as measured by either virus yield or quantitation of
viral RNA in infected cells. Increased cytokine mRNA levels at
this later time point could be due to a direct effect of the virus or
viral proteins on transcription or an indirect effect mediated by
double-stranded RNA intermediates formed during virus replication (30, 31).
The presence of a second increase in mRNA 24 h postinfection
also suggested the possibility of a secondary stimulus. Neutralizing
Abs to IL-1 (␣ and/or ␤) have been shown to inhibit HRV-14induced ICAM-1 up-regulation in human tracheal epithelial cells
and to inhibit IL-6 production in RSV-infected A549 cells. This
suggests that virus infection of epithelial cells could indirectly upregulate surface ICAM-1 expression or cytokine release via induction of IL-1 (12, 22). However, neutralizing Abs to IL-1␣, IL-1␤
or TNF had no effect on the overall levels of IL-8 and IL-6
achieved in response to HRV-39 infection in our experiments.
Signal transduction pathways responsible for rhinovirus-induced cytokine induction have yet to be elucidated. Several recent
studies have demonstrated a role for p38 kinase in viral-host interactions. The herpes simplex virus type 1 transactivator protein,
VP16, activates both JNK and p38 kinase, with maximum stimulation of p38 kinase occurring 9 h postinfection (32). The EBVencoded latent membrane protein 1 was shown to regulate IL-6
and IL-8 production through activation of p38 kinase (33). A role
for MAP kinases in respiratory virus infection has recently been
demonstrated in a study by Kujime et al. (34) in which p38 MAP
kinase and c-Jun-NH2-terminal kinase were shown to regulate
RANTES production in influenza virus-infected bronchial epithelial cells. In addition, Chen et al. (35) demonstrated that IL-8 production by RSV-infected A549 cells was linked to activation of
ERK2 kinase.
In this study we demonstrated that the p38 MAP kinase signal
transduction pathway plays an important role in HRV-induced cytokine biosynthesis in BEAS-2B cells. Activation of MAP kinases
can be induced by early stages of viral infection such as viral
binding and internalization as was shown with simian immunodeficiency virus activation of ERK1/2, p38 kinase, and JNK as well
as ERK2 activation by RSV infection of A549 cells (35, 36). Alternatively, MAP kinase activation can require viral protein synthesis or replication as was demonstrated with herpes simplex virus or EBV activation of JNK and p38 kinase (32, 33). In our
studies, short exposure (5 min) of cells to HRV resulted in phosphorylation of p38 kinase in a virus dose-dependent manner. The
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FIGURE 7. IL-8 mRNA expression in BEAS-2B cells infected with
HRV-39 and cultured in the presence or absence of 3 ␮M SB 239063.
Results are expressed as increase in mRNA levels as compared with untreated uninfected cells after normalization to GAPDH. Results are representative of three experiments.
p38 KINASE ACTIVATION BY RHINOVIRUS
The Journal of Immunology
thereby interrupting steady-state regulation. At later time points,
there was an increase in activity of an enzyme known to phosphorylate p38 kinase, MKK3/6. Although unlikely that an RNA
virus would have evolved similar complicated mechanisms for
host/cell interactions, a dual mechanism of p38 kinase activation
by rhinovirus is possible. Additional kinetic studies of both p38
kinase activation and inhibitor addition will provide insight into
the mechanism by which p38 kinase modulates HRV-induced cytokine production in BEAS-2B cells.
In summary, we have demonstrated that multiple cytokines
(GM-CSF and G-CSF) and CXC chemokines (IL-8, ENA-78, and
GRO␣) that specifically support neutrophil proliferation, infiltration, and activation are produced by BEAS-2B bronchial epithelial
cells in response to rhinovirus infection. The data suggest that
these mediators may collectively be responsible for the early neutrophil infiltration and activation associated with rhinovirus infection. In addition, these studies identified a role for the p38 kinase
signal transduction pathway in rhinovirus-induced cytokine elaboration. These results suggest that inhibition of p38 kinase may be
a new strategy for the treatment of neutrophil-mediated pathogenesis and inflammation associated with rhinovirus infection.
Acknowledgments
We acknowledge the enthusiastic technical assistance of Lauren Kaskiel.
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These results differed from the delayed activation of p38 kinase
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p38 KINASE ACTIVATION BY RHINOVIRUS