Divisions of 1 Pulmonary Medicine and 2 Immunologic and Infectious Diseases, Joseph Stokes, Jr. Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important interplay exists between specific
viral respiratory pathogens, most commonly rhinovirus (RV), and altered
airway responsiveness in the development and exacerbations of asthma. Given that RV infection reportedly induces the release of various cytokines in different cell types and that the reported effects of RV
on airway smooth muscle (ASM) responsiveness are highly comparable to
those obtained in ASM exposed to the proinflammatory cytokine
interleukin (IL)-1, this study examined whether RV (serotype 16)-mediated pertubations in ASM responsiveness are mechanistically coupled to altered induced expression and action of IL-1
in
RV-exposed isolated rabbit and human ASM tissue and cultured cells.
Relative to control tissues, ASM inoculated with RV exhibited
significantly increased maximal isometric contractility to ACh
(P < 0.01) and attenuated relaxation
to isoproterenol (P < 0.005). In
extended studies, we found that 1)
the RV-induced changes in ASM responsiveness were ablated by
pretreating the tissues with the IL-1 recombinant human receptor
antagonist; 2) in contrast to their
respective controls, RV-inoculated ASM tissue and cultured cells
exhibited progressively induced expression of IL-1
mRNA and
elaboration of IL-1
protein at 6 and 24 h after viral exposure; and
3) the latter effect of RV was
inhibited in the presence of a monoclonal antibody to intercellular
adhesion molecule-1, the endogenous receptor for most RV. Collectively,
these observations provide new evidence demonstrating that
"pro-asthmatic-like" pertubations in agonist responsiveness
elicited in RV-exposed ASM are largely attributed to the induced
autologous expression and autocrine action of IL-1
in the
virus-infected ASM.
cholinergic contractility; -adrenoceptor relaxation; viral
respiratory infection; cytokine signaling; airway reactivity; asthma
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IT IS CLINICALLY WELL established that rhinovirus (RV)
infection is the most common trigger of respiratory exacerbations in individuals with asthma (2, 9, 10, 21, 29). Moreover, a transient
airway hyperreactivity is also reported to occur in nonasthmatic
subjects after respiratory infections with RV (1, 9). Although the
basic mechanism(s) underlying RV-induced asthma exacerbation or
transient airway hyperreactivity remains to be established, there is
ample evidence that infection of various cell types with RV (e.g.,
respiratory epithelium, fibroblasts, monocytes, and macrophages)
induces the expression and release of different cytokines, including
interleukin (IL)-1, IL-6, IL-8, IL-9, IL-11, and tumor necrosis
factor (TNF)-
(19, 30, 32, 37). These findings suggest that specific
cytokines may play an important role in the pathogenesis of symptomatic
RV infections. In support of this concept, we recently demonstrated
that exposure of airway smooth muscle (ASM) tissue to specific
proinflammatory cytokines, most notably IL-1
, produces changes in
the tissue's responsiveness that closely resemble the characteristic
"proasthmatic" ASM phenotype, including heightened ASM
constrictor responsiveness and attenuated ASM relaxation responsiveness
to specific receptor/G protein-coupled agonists (16). Moreover,
comparable changes in responsiveness were observed in ASM tissues
passively sensitized with human atopic asthmatic serum, wherein the
latter changes were attributed to the autologously induced upregulated
expression, extracellular release, and autocrine action of IL-1
by
the ASM itself (17, 18). In light of this evidence, the present study examined whether infection of ASM with RV elicits changes in
constrictor and relaxant responsiveness that are mechanistically
coupled to the induced expression and autocrine action of IL-1
. The
results provide new evidence that 1)
RV exposure of isolated ASM produces heightened contractility to ACh
and attenuated relaxation to
-adrenergic receptor stimulation with
isoproterenol; 2) these effects are largely attributed to RV-induced autologous expression of IL-1
and
its resultant autocrine action in the RV-infected ASM; and 3) the autocrine release and effect
of IL-1
in the RV-exposed state is triggered by the binding of RV to
its endogenous receptor, intercellular adhesion molecule (ICAM)-1.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Adult New Zealand White rabbits were used in this study, which was approved by the Biosafety and Animal Research Committee of the Joseph Stokes Research Institute at Children's Hospital of Philadelphia. The animals had no signs of respiratory disease for several weeks before the study.
Culture of respiratory viruses. Virus
stock solutions were generated by infecting monolayer cultures of
sensitive cell systems with freshly isolated respiratory viruses from
the Clinical Virology Laboratory at the Children's Hospital of
Philadelphia, including human RV serotype 16 (RV16) and adenovirus.
Human embryonic lung fibroblasts (MRC-5) were used to culture the RV,
and human carcinoma cells from lung (A549) were used to culture
adenovirus. The cultures were grown in modified MEM supplemented with
Earle's balanced salt solution, 1% L-glutamine, 7.5%
FBS, HEPES buffer, and antimicrobial agents (5 µg/ml gentamicin, 10 µg/ml vancomycin, and 10 µg/ml amphotericin B). When infection was
notably advanced, as evidenced by obvious cytopathic effects (CPE),
cell supernatants were harvested and frozen in aliquots (~2-4 × 106 virus
particles/aliquot) at 70°C, as previously described (14), for use in the experiments described below.
Detection of respiratory viruses in ASM cells. Rabbit ASM cells cultured in our laboratory (see below) were propagated in 16 × 125-mm sterile plastic tissue culture dishes using the above complete medium. Confluent monolayers of ASM cells were then separately inoculated with stock solutions (~1 × 106 virus particles/T25 flask) of the above viruses as previously described in the laboratory. Detection of infection by adenovirus was confirmed by immunofluorescence staining of acetone-fixed infected cells using mouse monoclonal antibodies directed against adenovirus and a goat anti-mouse FITC conjugate. Due to the lack of commercially available antibodies to RV serotypes, detection of infection of the ASM cells with RV was confirmed by RT-PCR and Southern blotting. The PCRs were performed using cDNA reverse transcribed from total RNA isolated from ASM cells that were inoculated for 7 days with RV, using RV-specific primer pairs (i.e., 5' primer 5'-GCACTTCTGTTTCCCC-3' and 3' primer 5'-CGGACACCCAAAGTAG-3') and a cDNA probe prepared by pooling, purifying, and sequencing the above PCR reactions.
Viral inoculation of ASM tissue. Our
method for preparing rabbit ASM tissue has been previously described
(17). Briefly, after anesthesia with xylazine (10 mg/kg) and ketamine
(50 mg/kg), the animals were killed with an intravenous overdose of
pentobarbital sodium (130 mg/kg). The tracheae were removed, scraped of
loose connective tissue and epithelium, and then divided into 6- to 8-mm-ring segments. In comparable studies, human bronchial smooth muscle tissue was also obtained from patients undergoing lung resection
for peripheral lung carcinoma and having no evidence of obstructive
lung disease as assessed by routine pulmonary function testing
preoperatively. After lung resection, the specimens were macroscopically examined and, from tumor-free sites, bronchi (3-5 mm ID) from the 3rd to 5th generations were dissected free from the
surrounding parenchyma and sectioned into ring segments. Each alternate
adjacent human or rabbit ASM ring segment was then incubated for 90 min
at the optimal replication temperature for each virus in DMEM in the
absence and presence of maximum effective concentrations of either RV
or adenovirus (~1 × 106
viral particles/ml). The tissues were then further incubated for 24 h
at room temperature. The viral inoculation experiments were conducted
in the absence and presence of 1 h of pretreatment with the recombinant
human IL-1 receptor antagonist (IL-1ra; 140 ng/ml). The tissues were
aerated with a supplemental O2
mixture (95% O2-5%
CO2) throughout the incubation
period, and, thereafter, the tissue responsiveness to specific ASM
constrictor and relaxant agonists was compared as described below. In
extended experiments, IL-1 mRNA and protein expression were examined
in the human ASM tissue samples that were inoculated with RV16 or
vehicle alone in the absence and presence of anti-ICAM-1 monoclonal
antibody (MAb; 4 µg/ml) as described below.
Viral inoculation of cultured ASM
cells. Rabbit ASM cells cultured in our laboratory have
been previously characterized in detail with respect to their
distinguishing morphological, histological, and immunological features
(23). The cell isolation and subcultivation procedures were previously
described (23). Briefly, ASM cells were isolated from
epithelium-denuded trachealis muscle from adult New Zealand White
rabbits. After digestion in Ham's F-12 containing 30 µg/ml protease,
55 µg/ml type IV collagenase, and 100 µg/ml trypsin inhibitor, the
dissociated cells were centrifuged and resuspended in Ham's F-12
containing 10% FBS and 100 µg/ml gentamicin sulfate. The cells were
then inoculated in 100-mm tissue culture dishes, and, after 4 wk, the
cells had sufficiently proliferated to permit routine subcultivations.
At weekly intervals, the subcultivated cells were suspended and then
inoculated at a density of 1 × 104
cells/cm2 in
75-cm2 tissue culture flasks
containing Ham's F-12 with 10% FBS and were incubated at 37°C in
a humidified atmosphere of 5%
CO2-95% air. When the cells were
>90% confluent, the original culture medium was replaced with Ham's
F-12 for 24 h. The medium was then aspirated, and the cells
were inoculated, in separate experiments, with maximum effective
concentrations of either RV or adenovirus (~1 × 106 virus
particles/T25 flask) in 0.5 ml of
culture medium for 60 min at the optimal replication temperature for
each virus in the absence and presence of anti-ICAM-1 MAb (4 µg/ml).
The cells were then washed one time with fresh medium and incubated at
37°C for various time points. The cells were then prepared for
detection of IL-1 mRNA and protein expression as described below.
In parallel experiments, human bronchial smooth muscle cells
(Clonetics, San Diego, CA) derived from two male donors without lung
disease, aged 16 and 21 yr, were grown in smooth muscle basal medium
(SMBM) supplemented with 5% FBS, 5 ng/ml insulin, 10 ng/ml epidermal
growth factor, 2 ng/ml fibroblast growth factor, 50 ng/ml gentamicin,
and 50 ng/ml amphotericin B. The standard experimental protocol
involved growing the cells to confluence in the above medium and then
starving the cells in unsupplemented SMBM for 24 h, at which
time the cells were treated with either RV or adenovirus in the absence
and presence of anti-ICAM-1 MAb. The cellular RNA was harvested at the
various time points and used for detection of IL-1 mRNA, and the
cell medium was salvaged for determination of IL-1
protein release
as described below.
Pharmacodynamic measurements of ASM
responsiveness. After incubation of the tissue
preparations, each rabbit airway segment was suspended longitudinally
between stainless steel triangular supports in siliconized Harvard
20-ml organ baths. The lower support was secured to the base of the
organ bath, and the upper support was attached via a gold chain to a
Grass FT.03C force transducer from which isometric tension was
continuously displayed on a multichannel recorder. Care was taken to
place the membranous portion of the trachea between the supports to
maximize the recorded tension generated by the contracting trachealis
muscle. The tissues were bathed in modified Krebs-Ringer solution
containing (in mM) 125 NaCl, 14 NaHCO3, 4 KCl, 2.25 CaCl2 · H2O,
1.46 MgSO4 · H2O,
1.2 NaH2PO4 · H2O,
and 11 glucose. The baths were aerated with 5% CO2 in
O2; a pH of 7.35-7.40 was
maintained, and the organ bath temperature was held at 37°C.
Passive resting tension of each tracheal smooth muscle (TSM) segment
was set at 2.0 g after the tissue had been passively stretched to a
tension of 8 g to optimize the resting length of each segment as
previously described in our laboratory (14). The tissues were allowed
to equilibrate in the organ baths for 45 min, at which time each tissue
was primed with a 1-min exposure to
104 M ACh. Cholinergic
contractility was subsequently assessed in the TSM segments by
cumulative administration of ACh in final bath concentrations ranging
from 10
10 to
10
3 M. Thereafter, in
separate studies, relaxation dose-response curves to isoproterenol
(10
10 to
10
4 M) were conducted in
tissues half-maximally contracted with ACh. The relaxant responses to
isoproterenol were analyzed in terms of percent maximal relaxation
(Rmax) from the initial level of active cholinergic contraction, and sensitivity to the relaxing agent
was determined as the negative logarithm of the dose of isoproterenol
producing 50% of Rmax
(pD50; i.e., geometric mean ED50 value).
Determination of IL-1 expression in
ASM cells by RT-PCR and Southern blot analysis. Total
RNA was isolated from rabbit and human ASM cells in the absence and
presence of exposure of the cells to RV16 for varying durations using
the modified acid guanidinium thiocyanate phenol-chloroform extraction
method to include proteinase K (in 0.5% SDS) digestion of protein in
the initial RNA pellet (15). The concentration of each RNA sample was
then determined spectrophotometrically. This procedure consistently
yielded 15-25 µg of intact RNA for each
T75 flask of ASM cells. To analyze
the mRNA expression of IL-1
, we used an RT-PCR protocol and IL-1
primer pairs based on the published sequences of the rabbit (3) and
human (6) IL-1
genes and included the following primer sets:
1) rabbit-specific
5'-GCACCTCTCAGACAGAGTAC-3' and
5'-GTGGTTGCTGATAGAAGCTG-3' and
2) human-specific 5'-AGATGAAG
TGCTCCTTCCAG-3' and 5'-CAACACGCAGGACAGGTACAG-3'. To control for the transcription levels of the samples, we used 1) the rabbit-specific
-actin
primers 5'-CGACATCAAGGAGAAGCTG-3' and
5'-CTAGAAGCATTTGCGGTGC-3' and
2) the human-specific ribosomal protein L7 (RPL7) primers 5'-AAGAGGCTCTCATTTTCCTGGCTG-3'
and 5'-TCCGTTCCTCCCCATAATGTTCC-3', based on the
published sequences of the rabbit
-actin (25) and human RPL7 (28)
genes. cDNA was synthesized using 2.5 µg of total RNA isolated from
cells after 0, 6, and 24 h exposure to RV or medium alone. The cDNA was
primed with oligo(dT)12-18, and 2 µl of cDNA were used for each PCR. The cycling profile used was
as follows: denaturation, 95°C for 1 min; annealing, 52°C for
1.0 min, and extension, 72°C for 1 min with 35 cycles for the human
and rabbit IL-1
gene and 24 cycles for the
-actin and RPL7 genes.
The number of cycles was determined to be within the linear range of
the PCR products. Equal aliquots of each PCR reaction were then run on
a 1.2% agarose gel and subsequently transferred to a zeta probe
membrane overnight in 0.4 N NaOH. After capillary transfer, the DNA was
immobilized by ultraviolet (UV) cross-linking using a Stratalinker UV
cross-linker 2400 at 120,000 µJ/cm2 (Stratagene).
Prehybridization in a Techne hybridization oven was conducted for
2-3 h at 42°C in 50% formaldehyde, 7% (wt/vol) SDS,
0.25 M NaCl, 0.12 M
Na2HPO4
(pH 7.2), and 1 mM EDTA. Hybridization was for 20 h at 42°C in the
same solution. The IL-1
,
-actin, and RPL7 DNA levels were assayed
by Southern blot analysis using 32P-labeled probes, including the
rabbit-specific 358-bp IL-1
and 415-bp
-actin and human-specific
471-bp IL-1
and 157-bp RPL7 probes,
32P-labeled in random primer
reactions. The probes were prepared by pooling several RT-PCRs for the
PCR fragments, purifying them from a 1.2% agarose gel using a Qiaex II
agarose gel extraction kit, and then sequencing. Washes were as
follows: 1 × 15 min in 2× saline-sodium citrate (SSC),
0.1% SDS; 1 × 15 min in 0.1× SSC-0.1% SDS both at room
temperature; and 2 × 15 min at 50°C in 0.1× SSC-0.1% SDS. Southern blots were quantified by direct measurements of radioactivity in each band using a phosphorimager (Molecular Dynamics).
Determination of IL-1 expression in
human ASM cells by Northern blot analysis. Total RNA
isolated from the cultured human ASM cells maintained in SMBM in the
absence and presence of 0, 6, and 24 h exposure to RV was also
fractionated (15 µg/lane) in 1% agarose and 2.2 M formaldehyde
denaturing gels. After capillary transfer to zeta probe membranes
(Bio-Rad) in 10× SSC (1× SSC = 0.015 M sodium citrate), RNA
was immobilized by UV cross-linking using a Stratalinker UV
cross-linker 2400 at 120,000 µJ/cm2. After prehybridization
and hybridization, the IL-1
mRNA levels were examined by Northern
blot analysis using the human-specific IL-1
probe as described
above. A human-specific probe for constitutively expressed
glyceraldehyde-3-phosphate dehydrogenase was used as a control for RNA loading.
ELISA measurements of IL-1
protein. IL-1
protein levels were measured in the
culture medium from human ASM tissues and ASM cultured cells after 0, 6, and 24 h exposure to RV16 by means of a commercially available
enzyme-specific immunoassay. As per the manufacturer's protocol, the
assay was performed using a double-antibody sandwich strategy in which
an ACh esterase (AChE), Fab-conjugated IL-1
-specific secondary
antibody, is targeted to a first cytokine-captured antibody. The
enzymatic activity of the AChE was measured spectrophotometrically, and, relative to a linear standard curve (range: 0-250 pg/ml), the
results were used to quantify the amount of the targeted IL-1
present in the cell and tissue culture media.
Statistical analysis. Unless otherwise indicated, results are expressed as mean values ± SE. Statistical analysis was performed by means of the two-tailed paired Student's t-test. P values <0.05 were considered significant.
Reagents. The human IL-1, rabbit
-actin, and RV-specific primers were obtained from Integrated DNA
Technologies (Coralville, IA). The IL-1
ELISA kit and IL-1ra were
obtained from R&D Systems (Minneapolis, MN). The ICAM-1 MAb, ACh, and
isoproterenol hydrochloride were obtained from Sigma Chemical (St.
Louis, MO). All drug concentrations are expressed as final bath
concentrations. Isoproterenol and ACh were made fresh for each
experiment, dissolved in normal saline to prepare
10
4 and
10
3 M solutions,
respectively. The human tissue was provided by the Cooperative Human
Tissue Network, which is funded by the National Cancer Institute.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RV-induced changes in ASM
responsiveness. Detection of infection of ASM cells by
adenovirus was confirmed by evidence of CPE on days
3, 7, and
14 postinoculation. CPE were detected
using immunofluorescence staining of acetone-fixed infected cells with a mouse MAb directed against adenovirus and a goat anti-mouse FITC
conjugate (Fig.
1A).
Due to the lack of commercially available antibodies to RV serotypes,
as depicted in Fig. 1B, infection of
ASM cells with RV16 was confirmed by RT-PCR and Southern blotting using
RV-specific primer pairs (see MATERIALS AND METHODS) and a
cDNA probe prepared by purifying and sequencing the above PCRs.
|
To determine the role of IL-1 in mediating any induced changes in
ASM responsiveness in the RV-infected state, airway constrictor and
relaxation responses were separately examined in isolated TSM segments
that were exposed to either RV16 or vehicle alone in the absence and
presence of the endogenous IL-1ra. In contrast to adenovirus
inoculation, which had no effect relative to control tissues incubated
with vehicle alone, the maximal constrictor (Tmax) responses to ACh were
significantly enhanced in TSM that were exposed to RV16 (Fig.
2). Accordingly, the mean ± SE
Tmax values amounted to 99.08 ± 7.4 and 124.69 ± 21.00 g/g TSM wt in the control and
RV-exposed tissues, respectively (P < 0.01); and the corresponding
pD50 (i.e.,
log
ED50) values averaged 4.70 ± 0.08 and 5.13 ± 0.15
log M, respectively
(P < 0.05). This induced augmented
constrictor responsiveness to ACh, however, was largely prevented in
RV-exposed tissues that were pretreated with IL-1ra (Fig.
2), wherein the mean ± SE
Tmax and
pD50 values amounted to 108.58 ± 15.39 g/g TSM wt and 4.89 ± 0.12
log M, respectively. There were no significant differences between the latter
Tmax and
pD50 values obtained in the
IL-1ra-pretreated ASM and the corresponding determinations obtained in
control tissues, although the values were significantly different
(P < 0.05) from those obtained in
the presence of RV alone. Moreover, in contrast to RV-exposed ASM,
pretreatment with IL-1ra had no effect in adenovirus-exposed or control
(vehicle-treated) ASM tissues (data not shown).
|
In separate studies, during comparable levels of initial sustained
ACh-induced contractions in RV-exposed and control airway segments,
averaging ~45% of Tmax,
administration of the -adrenergic receptor agonist isoproterenol
elicited cumulative dose-dependent relaxation of the precontracted TSM
segments. Relative to control TSM, however,
Rmax and
pD50 values to isoproterenol were
significantly attenuated in the RV-exposed TSM (Fig.
3). Accordingly, the mean Rmax values for isoproterenol
amounted to 56.19 ± 7.50% in the RV-exposed tissues compared with
73.03 ± 4.10% in the control TSM
(P < 0.05); and the corresponding
pD50 values averaged 6.28 ± 0.08 and 6.59 ± 0.16
log M, respectively
(P < 0.05). Furthermore, as also
depicted in Fig. 3, the attenuated isoproterenol-induced relaxation
responses were largely ablated in RV-exposed TSM that were pretreated
with IL-1ra, wherein the mean Rmax
value for isoproterenol amounted to 73.64 ± 5.68% and the
corresponding pD50 value averaged 6.51 ± 0.13
log M. There were no significant differences
between the latter Rmax and
pD50 values and those obtained in
control TSM, although the values were significantly different
(P < 0.05) from those obtained in
the presence of RV alone. Finally, in contrast to RV, exposure of ASM
to adenovirus had no effect on the tissue's relaxant responsiveness to
isoproterenol (data not shown).
|
IL-1 mRNA expression in RV-exposed
ASM. Respiratory infections with RV have been
associated with the induced expression and elaboration of specific
cytokines in both natural and experimental human and animal studies
involving RV infection (19, 30, 32, 37). Given our above findings, and
to the extent that we recently identified a critical role for IL-1
in mediating changes in agonist responsiveness in atopic asthmatic
serum-sensitized ASM that are similar to the above effects obtained
after RV inoculation, we next examined whether exposure of ASM cells to
RV16 is associated with an induced altered expression and action of
IL-1
. Accordingly, in initial studies, using RT-PCR and
rabbit-specific IL-1
primers, cDNA was reverse transcribed from
total RNA isolated from cultured rabbit or human ASM cells and primed
with oligo(dT), and Southern blots were probed with cDNA probes
specific for rabbit and human IL-1
(see MATERIALS AND
METHODS). A 415-bp
-actin or 157-bp RPL7 probe
was also used to control for gel loading, and the signals for the
IL-1
,
-actin, or RPL7 PCR products were quantified using a
phosphorimager. As demonstrated in Fig. 4,
in contrast to the unaltered constitutive expression of
-actin or
RPL7, the IL-1
signal was significantly enhanced in both the rabbit
(Fig. 4A) and human (Fig.
4B) ASM cells that were exposed to
RV at 6 and 24 h, whereas the corresponding expression of IL-1
mRNA
was essentially unaltered in control cells that were treated with
vehicle alone. Qualitatively similar results were also obtained by
Northern blot analysis of IL-1
mRNA expression in cultured human ASM
cells that were exposed to RV compared with vehicle alone (data not shown). Finally, unlike IL-1
mRNA, we found no evidence of either constitutive or induced expression of TNF-
in either rabbit or human
ASM cells under control or RV-exposed conditions (data not shown).
|
IL-1 protein release by RV-exposed
ASM. In light of the above observations, additional
experiments were conducted to evaluate the effects of RV exposure of
human ASM cells and ASM tissue on the release of IL-1
protein in the
cell/tissue culture medium, examined using an IL-1
-specific
immunoassay. There were virtually undetectable levels of IL-1
in the
culture medium of either the ASM tissue or ASM cultured cell
preparations exposed to vehicle alone. In contrast to control
conditions, as exemplified in Fig. 5, ASM
cells exposed to RV depicted progressively enhanced elaboration of
IL-1
protein in the culture medium for up to 24 h. A
qualitatively similar effect of RV was obtained in the ASM tissue
preparations. Moreover, as depicted in Fig.
6, the stimulated release of IL-1
protein obtained at 24 h in the RV-exposed ASM tissue (Fig.
6A) and cultured ASM cells (Fig.
6B) was markedly inhibited by
pretreating the tissue or cell preparations with a monoclonal blocking
antibody to ICAM-1, the endogenous receptor for the vast majority of RV (13, 35). Thus, taken together, the above results demonstrate that
exposure of ASM tissue or cultured ASM cells to RV induces the mRNA
expression and elaboration of IL-1
protein in the RV-infected state,
a phenomenon attributed to binding of RV to its ICAM-1 receptor.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Viral respiratory infections are often associated with acute episodes
of wheezing and asthma exacerbations throughout childhood. Beyond
infancy, RV represents the predominant common cold virus that has been
implicated in triggering acute wheezing in asthmatic children and
adults. In this connection, RV infection has been associated with
increases in constrictor agonist-mediated airway reactivity (1, 2, 10,
21), increased influx of eosinophil and other inflammatory cells in the
respiratory tract (9), and the synthesis and elaboration of a host of
cytokines in both natural and experimental human and animal studies
involving RV infection (7, 19, 30, 33). A priori, when taken together, these effects of RV infection closely parallel the known
proinflammatory changes in airway function observed in atopic asthma
and, hence, suggest that a potentially important common mechanism
underlies the perturbed airway function seen both in asthma and after
RV infection. In this regard, it is relevant to note that we recently identified that the perturbations in ASM constrictor and relaxant responsiveness experimentally induced in naive ASM after its passive sensitization with human atopic asthmatic serum were attributed to the
autocrine release and action of IL-1 by the sensitized ASM itself
(16). Given the latter, the present study examined the potential role
of IL-1
in also mediating RV-induced perturbations in ASM
responsiveness. Our results demonstrated that
1) RV-exposed ASM displays
heightened ASM contractility to ACh and impaired relaxation
responsiveness to
-adrenoceptor stimulation;
2) these changes in ASM
responsiveness obtained in RV-exposed ASM are prevented by blockade of
IL-1
action; 3) exposure of ASM
tissue or cultured cells to RV induces the autologous mRNA expression
and elaboration of IL-1
protein by the ASM; and
4) the latter is attributed to the
binding of RV to its endogenous receptor, ICAM-1. Collectively, these
observations provide new evidence that the altered responsiveness of
RV-exposed ASM is largely due to the induced expression of IL-1
by
the virally infected ASM and that the latter cytokine acts in an
autocrine fashion to elicit enhanced ASM constrictor responsiveness and
impaired ASM relaxation.
Our above present findings closely resemble those recently reported in
isolated rabbit ASM passively sensitized with human atopic asthmatic
serum, wherein we found that comparable changes in ASM responsiveness
(i.e., increased contractility and attenuated relaxation) were
mechanistically coupled to the induced expression and autocrine action
of IL-1 protein in the atopic sensitized ASM (18). Moreover, in this
regard, the latter IL-1
-mediated changes in ASM responsiveness
obtained in atopic asthmatic serum-sensitized tissues (18), as well as
in naive ASM treated with exogenously administered IL-1
(16), were
found to be due to IL-1
-induced enhanced expression and function of
Gi protein (16, 17). The latter is
associated with inhibition of adenylate cyclase activity and, to a
lesser extent, with stimulation of phospholipase C (8, 27).
Accordingly, these Gi
protein-mediated effects result in both attenuated
-adrenoceptor-coupled ASM relaxation, secondary to reduced
accumulation of cAMP, and increased constrictor agonist-coupled ASM
contractility, secondary to enhanced accumulation of the
Ca2+-mobilizing second messenger
inositol 1,4,5-trisphosphate (26). In light of this evidence, it is
reasonable to assume that our observed IL-1
-mediated changes in ASM
responsiveness after RV exposure were likely related to IL-1
-induced
upregulated Gi protein effects on
receptor-coupled transmembrane signaling regulating ASM contraction and relaxation.
ICAM-1 is known to be the principal receptor for the vast majority (i.e., >90%) of RV subtypes (13, 35). Apart from its latter function, ICAM-1 activation has also been proposed in mediating the airway eosinophilia and hyperresponsiveness obtained in a primate model after antigen challenge because both the eosinophil influx and airway hyperresponsiveness were attenuated by the administration of an anti-ICAM-1 antibody (4, 15, 36). Whereas the latter study implicated a role for ICAM-1 activation that is coupled to eosinophil influx in mediating the altered airway responsiveness, a more recent study conducted in antigen-sensitized Brown Norway rats showed that administration of an anti-ICAM-1 antibody was found to attenuate the animal's airway constrictor hyperresponsiveness without producing a concomitant reduction in the degree of airway inflammation (31). Thus, although ICAM-1 activation may contribute to the development of airway constrictor hyperresponsiveness, the mechanism underlying this phenomenon may not be entirely dependent on inflammatory cell influx.
In extending the above concept, it is relevant to note that expression
of ICAM-1 has been demonstrated in lung stromal cells, including
endothelial cells, epithelial cells, and ASM cells, and that ICAM-1
expression in these cell types is upregulated after exposure of
asthmatic subjects to allergen (20, 22, 24, 33, 34). Moreover, recent
studies have demonstrated that different viral respiratory pathogens,
including RV, can directly infect lung stromal cells (e.g.,
fibroblasts) and thereby induce the elaboration of specific cytokines
(7). Collectively, this evidence, when considered in light of our
present finding that the administration of anti-ICAM-1 MAb to ASM
tissue and cultured cells largely inhibited RV-induced IL-1 release
(Fig. 6), suggests a critical role for RV/ICAM-1-coupled activation of
ASM in autologously mediating the observed changes in ASM
responsiveness, secondary to the enhanced release and autocrine action
of IL-1
. Thus the findings of the present study, together with the
above recent evidence, support the emerging general concept that,
notwithstanding the contemporary view that the effects of RV on airway
responsiveness likely occur secondary to infection of the airway
epithelium, the ASM itself constitutes an autologously regulated system
that, when directly activated by RV, can also induce the mRNA
expression and autocrine release and action of specific cytokines
(notably, IL-1
), which lead to perturbations in ASM responsiveness.
Thus, in agreement with previous studies on other resident stromal
cells in the airways, such as fibroblasts and epithelial cells (7, 10,
30), our present observations are consistent with the notion that,
under specific conditions (e.g., RV infection), ASM cells can also act
as important contributors to tissue inflammation and intrinsically
altered airway responsiveness.
Because our present observations in isolated ASM tissue and cultured ASM cells pertain to in vitro studies, the question is raised as to the relevance of these findings with respect to the in vivo state. Clearly, this fundamental question remains to be systematically addressed in appropriate in vivo studies, although our present observations provoke certain noteworthy considerations. Accordingly, in light of the evidence provided herein that implicates a role for direct RV infection of ASM in inducing its altered responsiveness, the compelling consideration is raised that RV-mediated changes in airway responsiveness in vivo may, at least in part, also be attributed to direct infection of the ASM by the viral pathogen. Such a process might involve extension of an initial RV infection of the airway epithelium into the adjacent ASM tissue. Alternatively, or possibly together with the latter mechanism, it is also conceivable that, in the presence of any RV-induced disruption of the integrity of the respiratory epithelium, infection by the virus of the underlying ASM may be facilitated. Another issue pertains to the potential role of ICAM-1 activation in vivo. In this connection, the present findings suggest that the effects of RV infection on ASM responsiveness are attributed to binding of the viral pathogen to its key endogenous receptor in ASM (i.e., ICAM-1). The latter also serves as the counterreceptor for the integrin lymphocyte function-associated antigen (LFA)-1, which is present on various leukocytes and is known to mediate a variety of cellular responses that are associated with altered local immune function. Given this evidence, the consideration is raised that, in addition to any potential direct coupling of RV to ICAM-I in ASM in the in vivo state, LFA-1-mediated activation of ICAM-1 in vivo may further serve to initiate and/or propagate the airway proinflammatory response to RV infection. The presence and relative contributions of the above potential in vivo mechanisms remain to be elucidated.
In conclusion, the present study examined the role and mechanism of
action of RV infection of isolated ASM in mediating changes in
agonist-induced ASM constrictor and relaxant responsiveness. The
results demonstrated that 1)
inoculation of ASM with RV induces enhanced ASM constrictor
responsiveness to ACh and impaired -adrenoceptor-mediated ASM
relaxation; 2) these changes in ASM
responsiveness are mechanistically associated with RV-induced enhanced
IL-1
mRNA and protein expression, eliciting an autocrine action in
ASM; and 3) the latter upregulated expression and autocrine action of IL-1
is triggered by the binding of RV to its endogenous receptor, ICAM-1. Given the well-established clinical significance of RV infection in the pathogenesis of altered airway reactivity and asthma exacerbation, the above findings identify
an important role and mechanism by which the ASM autologously induces
its state of altered responsiveness after infection with RV.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Grunstein, S. Chang, and S. Ling for expert technical assistance and Margaret Brown for assistance with typing the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported in part by the National Heart, Lung, and Blood Institute Grants HL-31467 and HL-58245, a Parker B. Francis Fellowship Award, and an Institutional Developmental Fund Award from the Joseph Stokes Jr. Research Institute of the Children's Hospital of Philadelphia.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. M. Grunstein, Division of Pulmonary Medicine, The Children's Hospital of Philadelphia, Univ. of Pennsylvania School of Medicine, 34th St. and Civic Center Blvd., Philadelphia, PA 19104.
Received 2 November 1998; accepted in final form 24 February 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Blair, H. T.,
S. B. Greenberg,
P. M. Stevens,
P. A. Bilunos,
and
R. B. Couch.
Effects of rhinovirus infection on pulmonary function of healthy human volunteers.
Am. Rev. Respir. Dis.
114:
95-102,
1976[Medline].
2.
Busse, W. W.
Respiratory infections: their role in airway responsiveness and the pathogenesis of asthma.
J. Allergy Clin. Immunol.
85:
671-683,
1990[Medline].
3.
Cannon, J. G.,
D. B. D. Clark,
P. Wingfield,
U. Schmeissner,
C. Losberger,
C. A. Dinarello,
and
A. R. Shaw.
Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia.
J. Immunol.
142:
2299-2306,
1989
4.
Chin, J. E.,
G. E. Winterrowd,
C. A. Hatfield,
and
J. R. Brashler.
Involvement of intercellular adhesion molecule-1 in the antigen induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation.
Am. J. Respir. Cell Mol. Biol.
18:
158-167,
1998
5.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-choloform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
6.
Conlon, P. J., III, D. J. Cosman, K. H. Grabstein, T. P. Hopp, S. R. Kronheim,
A. D. Larsen, C. J. March, V. L. Price,
and D. P. Cerretti (Inventors). Gene Encoding
Biologically Active Human Interleukin 1. US Patent 5,122,459. 16 June 1992.
7.
Einarsson, O.,
G. P. Geba,
Z. Zhu,
M. Landry,
and
J. A. Elias.
Interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness.
J. Clin. Invest.
97:
915-924,
1996
8.
Felder, C. C.,
H. L. Williams,
and
J. Axelrod.
A transduction pathway associated with receptors coupled to the inhibitory guanine nucleotide binding protein Gi that amplifies ATP-mediated arachidonic acid release.
Proc. Natl. Acad. Sci. USA
88:
6477-6480,
1991[Abstract].
9.
Fraenkel, D. J.,
P. G. Bardin,
G. Sanderson,
F. Lampe,
S. L. Johnston,
and
S. T. Holgate.
Lower airways inflammation during rhinovirus colds in normal and asthmatic subjects.
Am. J. Respir. Crit. Care Med.
151:
879-886,
1995[Abstract].
10.
Gern, J. E.,
and
W. W. Busse.
The effects of rhinovirus infections on allergic airway responses.
Am. J. Respir. Crit. Care Med.
152:
40-45,
1995.
11.
Gleaves, C. A.,
R. L. Hodinka,
S. L. G. Johnston,
and
E. M. Swierkosz.
Cumulative techniques and procedures in clinical microbiology
In: Laboratory Diagnosis of Viral Infections, edited by E. J. Baron. Washington, DC: Am. Soc. Microbiol., 1994, p. 1-35.
12.
Gosset, P.,
I. Tillie-Leblond,
A. Janin,
C. H. Marguette,
M. C. Copin,
B. Wallaert,
and
A. B. Tonnel.
Expression of E-selectin, ICAM-1 and VCAM-1 on bronchial biopsies from allergic and non-allergic asthmatic patients.
Int. Arch. Allergy Immunol.
106:
69-77,
1995[Medline].
13.
Greve, J. M.,
G. Davis,
A. M. Meyer,
C. P. Forte,
S. C. Yost,
C. W. Marlor,
M. E. Kamarck,
and
A. McClelland.
The major human rhinovirus receptor is ICAM-1.
Cell
56:
839-847,
1989[Medline].
14.
Grunstein, M. M.,
S. T. Chuang,
C. M. Schramm,
and
N. A. Pawlowski.
Role of endothelin-1 in regulating rabbit airway contractility.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L75-L82,
1991
15.
Gundel, R. H.,
C. D. Wegner,
C. A. Torcellini,
and
L. G. Letts.
The role of intercellular adhesion molecule-1 in chronic airway inflammation.
Clin. Exp. Allergy
22:
569-575,
1992[Medline].
16.
Hakonarson, H.,
D. J. Herrick,
P. Gonzalez-Serrano,
and
M. M. Grunstein.
Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle.
J. Clin. Invest.
97:
2593-2600,
1996
17.
Hakonarson, H.,
D. J. Herrick,
P. Gonzalez-Serrano,
and
M. M. Grunstein.
Autocrine role of IL-1i in altered responsiveness of atopic asthmatic sensitized airway smooth muscle.
J. Clin. Invest.
99:
117-124,
1997
18.
Hakonarson, H.,
D. J. Herrick,
and
M. M. Grunstein.
Mechanism of impaired -adrenoceptor responsiveness in atopic sensitized airway smooth muscle.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L645-L652,
1995
19.
Johnston, S. L.
Natural and experimental rhinovirus infections of the lower respiratory tract.
Am. J. Respir. Crit. Care Med.
152:
S46-S52,
1995[Medline].
20.
Lazaar, A. L.,
H. E. Reitz,
R. A. Panettieri, Jr.,
S. P. Peters,
and
E. Pure.
Antigen receptor-stimulated peripheral blood and bronchoalveolar lavage-derived T cells induce MHC class II and ICAM-1 expression on human airway smooth muscle.
Am. J. Respir. Cell Mol. Biol.
16:
38-45,
1997[Abstract].
21.
Lemanske, R. F.,
E. C. Dick,
C. A. Swenson,
R. F. Vrtis,
and
W. W. Busse.
Rhinovirus upper respiratory tract infection increases airway hyperreactivity and late asthmatic reactions.
J. Clin. Invest.
83:
1-10,
1989[Medline].
22.
Manolitsas, N. D.,
J. C. Trigg,
A. E. McAulay,
J. H. Wang,
S. E. Jordan,
A. J. D'Ardenne,
and
R. J. Davies.
The expression of intracellular adhesion molecule-1 and the B1-integrins in asthma.
Eur. Respir. J.
7:
1439-1444,
1994
23.
Noveral, J. P.,
and
M. M. Grunstein.
Role and mechanism of thromboxane-induced proliferation of cultured airway smooth muscle cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L555-L561,
1992
24.
Patel, J. A.,
M. Kunimoto,
T. C. Sim,
R. Garofalo,
T. Elliott,
S. Baron,
O. Ruuskanen,
T. Chonmaitree,
P. L. Ogra,
and
F. Schmalstieg.
Interleukin-1 mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus.
Am. J. Respir. Cell Mol. Biol.
13:
602-609,
1995[Abstract].
25.
Putney, S. D.,
W. C. Herlihy,
and
P. Schimmel.
A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing.
Nature
302:
718-721,
1983[Medline].
26.
Schramm, C. M.,
and
M. M. Grunstein.
Assessment of signal transduction mechanisms regulating airway smooth muscle contractility.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L119-L139,
1992
27.
Selbie, L. A.,
K. Darby,
C. Schmitz-Peiffer,
C. L. Browne,
H. Herzog,
J. Shine,
and
T. J. Biden.
Synergistic interaction of Y1-neuropeptide Y and alpha 1b-adrenergic receptors in the regulation of phospholipase C, protein kinase C, and arachidonic acid production.
J. Biol. Chem.
20:
11789-11796,
1995.
28.
Seshadri, T.,
J. A. Uzman,
J. Oshima,
and
J. Campisi.
Identification of a transcript that is down-regulated in senescent human fibroblasts. Cloning, sequence analysis, and regulation of the human L7 ribosomal protein gene.
J. Biol. Chem.
268:
18474-18480,
1993
29.
Stark, J. M.,
and
F. M. Graziano.
Lower airway responses to viruses.
In: Asthma and Rhinitis, edited by W. W. Busse,
and S. T. Holgate. Boston, MA: Blackwell Scientific, 1995, p. 1229-1243.
30.
Subauste, M. C.,
D. B. Jacoby,
S. M. Richards,
and
D. Proud.
Infection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure.
J. Clin. Invest.
96:
549-557,
1995[Medline].
31.
Sun, J.,
W. Elwood,
A. Haczku,
P. J. Barnes,
P. J. Hellewell,
P. G. Hellewell,
and
K. F. Chung.
Contribution of intercellular adhesion molecule-1 in allergen-induced airway hyperresponsiveness and inflammation in sensitized brown-norway rats.
Int. Arch. Allergy Immunol.
104:
291-295,
1994[Medline].
32.
Terajima, M.,
M. Yamaya,
K. Sekizawa,
S. Okinaga,
T. Suzuki,
M. Yamada,
K. Nakayama,
T. Ohrui,
T. Oshima,
Y. Numazaki,
and
H. Sasaki.
Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L749-L759,
1997
33.
Tosi, M. F.,
J. M. Stark,
A. Hamedani,
C. W. Smith,
D. C. Gruenert,
and
Y. T. Huang.
Intercellular adhesion molecule-1 (ICAM)-1-dependent and ICAM-1 independent adhesive interactions between polymorphonuclear leukocytes and human airway epithelial cells infected with parainfluenza virus type 2.
J. Immunol.
149:
3345-3349,
1992
34.
Tosi, M. F.,
J. M. Stark,
C. W. Smith,
A. Hamedani,
D. C. Gruenert,
and
M. D. Infeld.
Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion.
Am. J. Respir. Cell Mol. Biol.
7:
214-221,
1992[Medline].
35.
Uncapher, C. R.,
C. M. DeWitt,
and
R. J. Colonno.
The major and minor group receptor families contain all but one human rhinovirus serotype.
Virology
180:
814-817,
1991[Medline].
36.
Wegner, C. D.,
R. H. Gundel,
P. Reilly,
N. Haynes,
L. G. G. Letts,
and
R. Rothlein.
Intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma.
Science
247:
456-459,
1990[Medline].
37.
Zhu, Z.,
W. Tang,
J. M. Gwaltney, Jr.,
Y. Wu,
and
J. A. Elias.
Rhinovirus stimulation of interleukin-8 in vivo and in vitro: role of NF-B.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L814-L824,
1997[Medline].