Division of Pulmonary Medicine, Joseph Stokes, Jr. Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The airway responses to allergen exposure in allergic
asthma are qualitatively similar to those elicited by specific viral respiratory pathogens, most notably rhinovirus (RV), suggesting that
the altered airway responsiveness seen in allergic asthma and that
elicited by viral respiratory tract infection may share a common
underlying mechanism. To the extent that T helper cell type 2 (Th2)
cytokines have been implicated in the pathogenesis of allergic asthma,
this study examined the potential role(s) of Th2-type cytokines in
mediating pro-asthmatic-like changes in airway smooth muscle (ASM)
responsiveness after inoculation of naive ASM with human RV. Isolated
rabbit ASM tissues and cultured human ASM cells were exposed to RV
(serotype 16) for 24 h in the absence and presence of monoclonal
blocking antibodies (MAbs) or antagonists directed against either the
Th2-type cytokines interleukin (IL)-4 and IL-5, intercellular adhesion
molecule (ICAM)-1 (the endogenous host receptor for most RVs), or the
pleiotropic proinflammatory cytokine IL-1. Relative to control
(vehicle-treated) tissues, RV-exposed ASM exhibited significantly
enhanced isometric contractility to acetylcholine and impaired
relaxation to isoproterenol. These pro-asthmatic-like changes in ASM
responsiveness were ablated by pretreating the RV-exposed tissues with
either IL-5-receptor-
blocking antibody or human recombinant
IL-1-receptor antagonist, whereas IL-4 neutralizing antibody had no
effect. Extended studies further demonstrated that inoculation of ASM
cells with RV elicited 1) an increased mRNA expression and
release of IL-5 protein, which was inhibited in the presence of
anti-ICAM-1 MAb, and 2) an enhanced release of IL-1
protein,
which was inhibited in the presence of IL-5 receptor-
antibody.
Collectively, these observations provide new evidence demonstrating
that RV-induced changes in ASM responsiveness are largely attributed to
ICAM-1-dependent activation of a cooperative autocrine signaling
mechanism involving upregulated IL-5-mediated release of IL-1
by the
RV-exposed ASM itself.
T helper cell type 2 cytokines; airway smooth muscle; asthma
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INTRODUCTION |
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IN ASTHMATIC INDIVIDUALS, it is clinically well
recognized that infection of the respiratory tract with certain viral
pathogens often precipitates acute wheezing and symptom deterioration.
Among these viral agents, rhinovirus (RV), the principal pathogen
responsible for the common cold, represents the most frequent trigger
of exacerbation of asthma symptoms (8, 12, 13, 27). Moreover, in this connection, a transient airway hyperreactivity has also been documented in normal (i.e., nonasthmatic) individuals after respiratory tract infection with RV (7, 12). Although the basic mechanism(s) mediating
RV-induced exacerbation of asthma and airway hyperreactivity remains to
be identified, a host of studies (25, 32, 34, 37) with various cells
types (e.g., respiratory epithelium, fibroblasts, monocytes, and
macrophages) have demonstrated that infection with RV elicits the
expression and elaboration of various proinflammatory cytokines,
including interleukin (IL)-1, IL-6, IL-8, IL-9, IL-11, and tumor
necrosis factor (TNF)-
. It is generally believed that the actions of
these cytokines, individually or in combination, likely underlie the
clinical manifestation of symptomatic RV infection. In support of this
concept, we recently identified that, notwithstanding the
above-mentioned cell types, RV is also capable of directly infecting
airway smooth muscle (ASM) tissue and cultured ASM cells and that this
phenomenon is associated with the induction of
"pro-asthmatic-like" changes in ASM responsiveness, including
heightened constrictor agonist-mediated ASM contraction and impaired
-adrenoceptor-mediated ASM relaxation (17, 23). Both these
RV-induced changes in ASM responsiveness are qualitatively similar to
those obtained in atopic asthmatic serum-sensitized ASM (19, 20),
suggesting that the induced altered ASM responses in allergic asthma
and those elicited after specific viral respiratory infections (e.g.,
with RV) share certain common mechanistic pathways. Indeed, in support
of this concept, the collection of recent evidence from studies
independently examining mechanisms of altered ASM responsiveness in the
atopic asthmatic sensitized state (19) and after RV infection (17) of
isolated ASM demonstrates that both of these conditions are associated with an induced endogenous release and autocrine action of the pleiotropic cytokine IL-1
. In light of this evidence, together with
the wealth of information that implicates a crucial role of T helper
cell type 2 (Th2)-type cytokines (e.g., IL-4 and IL-5) in mediating the
pulmonary inflammatory response and airway hyperreactivity in allergic
asthma (1, 11, 24, 29, 36), the present study examined whether
RV-induced changes in ASM responsiveness are, at least in part,
mechanistically coupled to activation of the Th2-type cytokine
signaling pathways in the RV-infected state. The results provide
evidence demonstrating that 1) exposure of isolated ASM to RV
elicits increased contractility of ASM to acetylcholine (ACh) and
attenuated relaxation of the tissue to
-adrenoceptor stimulation
with isoproterenol; 2) these changes in ASM responsiveness are
associated with an induced upregulated mRNA expression and release of
IL-5 protein by the RV-exposed ASM; and 3) the latter autologous release of IL-5 acts in an autocrine fashion to mediate the
subsequent autologous release of IL-1
, which, in turn, is responsible for the RV-induced changes in ASM responsiveness. Collectively, these findings demonstrate that the effects of RV on
airway responsiveness are largely attributed to the induction of a
cooperative autocrine signaling mechanism that involves IL-5 and
IL-1
release and action in the RV-exposed ASM.
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METHODS |
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Animals. Twenty-one 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 (Philadelphia, PA). The animals had no signs of respiratory disease for several weeks before study.
Culture of human RV.
A stock solution of human RV serotype 16 (RV16) was prepared by
infecting monolayer cultures of human embryonic lung fibroblasts (MRC-5) with freshly isolated RV16 from the Clinical Virology Laboratory at the Children's Hospital of Philadelphia. As previously described (17, 23), the cultures were grown in modified MEM supplemented with Earle's balanced salt solution, 1%
L-glutamine, 7.5% fetal bovine serum, HEPES
buffer, and antimicrobial agents (5 µg/ml of gentamicin,
10 µg/ml of vancomycin, and 10 µg/ml of amphotericin B). When the
infection was notably advanced, as evidenced by cytopathic effects, the
cell supernatants were harvested and frozen in aliquots (~2-4 × 106 virus particles/aliquot) at 70°C for
use in the experiments described in RV inoculation of ASM
tissue.
RV inoculation of ASM tissue.
Our method for preparing rabbit ASM tissue has been previously
described (19, 20). In brief, 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. Each alternate adjacent ASM ring
segment was then incubated for 24 h at the optimal replication
temperature for human RV in Dulbecco's modified Eagle's medium in the
absence and presence of a maximal effective concentration of RV (~1 × 106 viral particles/ml) as previously reported by
our laboratory (17, 23). The inoculation experiments were conducted in
the absence and presence of 1 h of pretreatment of the tissues with either an IL-4 neutralizing monoclonal antibody (NAb; 150 ng/ml), an
IL-5-receptor- (IL-5R
) blocking antibody (50 ng/ml),
or 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 responsiveness of the tissues to specific
constrictor and relaxant agonists was compared as described in
Pharmacodynamic studies of ASM responsiveness.
Pharmacodynamic studies of ASM responsiveness.
After incubation of the tissue preparations, each ASM 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
oxygen, a pH of 7.35-7.40 was maintained, and the organ bath
temperature was held at 37°C. Passive resting tension of each ASM
segment was set at 1.5-2.0 g after the tissue had been passively
stretched to a tension of 8 g to optimize its resting length for
contraction as previously described by our laboratory (20). 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 ASM 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 initial constrictor
dose-response curves to ACh were analyzed in terms of the maximal
isometric contractile force (Tmax) of the tissues, and
sensitivity to the agonist is expressed as the negative logarithm of
the dose of ACh producing 50% of Tmax (pD50; i.e., geometric mean ED50 value). 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 corresponding pD50 value associated with 50% of
Rmax.
RV inoculation of cultured ASM cells.
Human bronchial smooth muscle cells (Clonetics, San Diego, CA) derived
from two male donors aged 16 and 21 yr were grown in smooth muscle
basal medium (SMBM) supplemented with 5% fetal bovine serum, 5 ng/ml
of insulin, 10 ng/ml of epidermal growth factor, 2 ng/ml of fibroblast
growth factor, 50 ng/ml of gentamicin, and 50 ng/ml of amphotericin B. The cells were fully characterized by the vendor with respect to the
expression of specific markers that confirmed their selective smooth
muscle phenotype and excluded any contamination with other cell types.
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 for
0, 3, 6, and 24 h with RV in the absence and presence of maximally
effective concentrations of IL-4 NAb, IL-5R blocking antibody, or a
monoclonal blocking antibody (MAb) to intercellular adhesion molecule
(ICAM)-1. The cells were then examined for mRNA and protein release of
IL-5 and IL-1
as described in Determination of IL-5 mRNA
expression in human ASM cells and ELISA measurements of IL-5
and IL-1
protein release.
Determination of IL-5 mRNA expression in human ASM cells. Total RNA was isolated from the ASM cell preparations with the modified guanidinium thiocyanate-phenol-chloroform extraction method to include proteinase K (in 5% SDS) for digestion of protein in the initial RNA pellet as previous described by our laboratory (17, 22). The concentration of each RNA sample was determined spectrophotometrically. This procedure consistently produced yields of 15-25 µg of intact RNA from each T-75 flask of cultured human ASM cells. To analyze for mRNA expression of IL-5, we used a RT-PCR protocol that included a human-specific primer for the cytokine as well as for the constitutively expressed ribosomal protein (RP) L7. cDNA was synthesized from total RNA isolated from ASM cells grown under control (untreated) and RV-inoculated conditions. The cDNA was primed with oligo(dT)12-18 and extended with Superscript II RT (GIBCO BRL). The PCR was used to amplify the specific products from each cDNA reaction based on the published sequences of the human IL-5 and RPL7 genes (9, 30) and included the primer sets 5'-GTATGCCATCCCCACAGAAA-3' (5'-primer; product is 433 bp) and 5'-TACAGACATTCACAGCCACC-3' (3'-primer) for IL-5 and 5'-AAGAGGCTCTCATTTTCCTGGCTG-3' (5'-primer; product is 157 bp) and 5'-TCCGTTCCTCCCCATAATGTTCC-3' (3'-primer) for RPL7.
The cycling profile used was as follows: denaturation at 95°C for 1 min, annealing at 52-55°C for 1 min, and extension at 72°C for 1 min for 34 cycles for the IL-5 gene and 26 cycles for the RPL7 gene. The number of cycles was determined to be in the linear range of the PCR products. The PCRs for the human IL-5 and RPL7 primers were performed with equivalent amounts of cDNA prepared from 2.5 µg of total RNA. Equal aliquots of each PCR 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 cross-linking with a Stratalinker UV Crosslinker 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-5 and RPL7 DNA levels were assayed by Southern blot analysis with 32P-labeled probes prepared by pooling several RT-PCRs for the individual IL-5 and RPL7 PCR fragments and purifying them from a 1.2% agarose gel with the Qiaex II agarose gel extraction kit. The individual PCR products were subsequently sequenced for confirmation. 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 quantitated by direct measurement of the radioactivity in each band with a PhosphorImager (Molecular Dynamics).ELISA measurements of IL-5 and IL-1 protein
release.
Both IL-5 and IL-1
protein levels were assayed in the culture medium
of ASM cells that were exposed for 0, 3, 6, and 24 h to vehicle alone
(control) or to RV in the absence and presence of anti-ICAM-1 MAb, IL-4
NAb, and IL-5R
antibody. The IL-5 and IL-1
protein levels were
quantitatively assessed with an enzyme-specific immunoassay as
previously described by our laboratory (19, 22). The latter assay was
performed with a double-antibody sandwich strategy in which an
acetylcholinesterase-Fab-conjugated IL-5 or IL-1
secondary antibody
is targeted to a first cytokine-captured antibody. The enzymatic
activity of acetylcholinesterase 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-5 and
IL-1
present in the cell culture medium.
Reagents.
The human ASM cells and SMBM were obtained from Clonetics (San Diego,
CA). The IL-5 and RPL7 primers were obtained from Integrated DNA
Technologies (Coralville, IA). IL-1ra, anti-human IL-5R antibody, IL-4 NAb, the IL-5 and IL-1
ELISA kits, the mouse anti-human IL-1
primary antibody, and the anti-mouse secondary antibody used in the
protein assay studies were purchased from R&D Systems (Minneapolis,
MN). ACh and isoproterenol were purchased from Sigma (St. Louis, MO).
All drug concentrations are expressed as final bath concentrations.
Isoproterenol and ACh were made fresh for each experiment and were
dissolved in normal saline to prepare 10
4 M stock solutions.
Statistical analysis. Unless otherwise indicated, the results are expressed as means ± SE. Statistical analysis was performed with two-tailed Student's t-test. P values < 0.05 were considered significant.
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RESULTS |
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Role of IL-5 and IL-1 in mediating RV-induced
changes in ASM responsiveness. In light of recent evidence
demonstrating that an induced autocrine interaction between IL-5 and
IL-1
mediates the altered responsiveness of ASM in the atopic
asthmatic sensitized state (21), studies were conducted to initially
evaluate the potential roles of the Th2-type cytokines IL-4 and IL-5 as
well as the pleiotropic proinflammatory cytokine IL-1
in regulating ASM responsiveness in the presence of RV inoculation. Agonist constrictor and relaxation responses were separately examined in
isolated rabbit ASM segments 24 h after inoculation of the tissues with
vehicle alone (control) or with RV16 in the absence and presence of
either IL-4 NAb, IL-5R
antibody, or IL-1ra (see METHODS). As depicted in Fig.
1A, relative to control tissues, the constrictor responses and sensitivities (pD50; i.e.,
log ED50 values) to exogenously administered ACh
were significantly increased in ASM exposed to RV. Accordingly, the
mean maximal constrictor response (Tmax) generated at the
highest administered concentration of ACh (10
3 M)
amounted to 101.9 ± 5.9 and 122.4 ± 8.5 g/g tracheal smooth muscle
(TSM) in the control and RV-exposed tissues, respectively (P < 0.01), representing an RV-induced relative increase in
Tmax of 20.1 ± 3.8% above the control value. Comparably,
the corresponding mean pD50 values amounted to 4.99 ± 0.08 and 5.45 ± 0.09
log M in the control and RV-exposed ASM,
respectively (P < 0.01). These induced enhanced ASM
constrictor responses to ACh were unaffected by pretreating RV-exposed
tissues with IL-4 NAb (data not shown), wherein the mean
Tmax and pD50 values remained elevated at 128.3 ± 9.2 g/g TSM and 5.51 ± 0.09
log M, respectively. In
contrast, the augmented constrictor responses to ACh were prevented in
RV-exposed tissues that were pretreated with a maximally effective
concentration (i.e., 50 ng/ml) of IL-5R
antibody (Fig. 1A).
In these tissues, the mean Tmax and pD50 values
amounted to 104.9 ± g/g TSM and 5.01 ± 0.08
log M,
respectively, and both these values were not significantly different
from those obtained in control ASM. Similarly, in comparable
experiments, consistent with earlier observations by Hakonarson et al.
(18), we found that pretreatment of RV-exposed ASM with
human recombinant IL-1ra (140 ng/ml) also ablated the heightened
constrictor responsiveness of the tissues to ACh (Fig. 1B).
Accordingly, the mean Tmax values in the RV-exposed and
control tissues amounted to 131.6 ± 10.7 and 104.3 ± 6.2 g/g TSM,
respectively (P < 0.01), and the corresponding
pD50 values averaged 5.17 ± 0.09 and 4.78 ± 0.09
log M, respectively (P < 0.05). In the presence of
IL-1ra, the Tmax and pD50 values in the
RV-exposed ASM were similar to those obtained in control tissues and
averaged 107.4 ± 5.1 g/g TSM and 4.83 ± 0.11
log M,
respectively. In contrast to these observations obtained in RV-exposed
ASM, neither IL-4 NAb, IL-5R
antibody, nor IL-1ra had any
appreciable effect on ASM contractility to ACh in control tissues (data
not shown).
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In separate studies during comparable levels of initial ACh-induced
contractions in control and RV-exposed ASM segments averaging ~45%
of Tmax, administration of the -adrenoceptor agonist
isoproterenol elicited cumulative dose-dependent relaxation of the
precontracted tissues. As depicted in Fig.
2A, relative to control ASM, the maximal relaxation (Rmax) responses and sensitivities
(pD50 values) to isoproterenol were significantly reduced
in the corresponding RV-exposed tissues. Accordingly, the mean
Rmax values in the RV-exposed and control ASM amounted to
47.5 ± 5.4 and 59.9 ± 7.2%, respectively (P < 0.01), and
the corresponding pD50 values for isoproterenol averaged
5.81 ± 0.11 and 6.19 ± 0.12
log M, respectively (P < 0.05). These attenuated relaxation responses to isoproterenol were
unaffected in RV-exposed tissues that were pretreated with IL-4 NAb
where their Rmax and pD50 values averaged 49.2 ± 7.1% and 5.83 ± 0.08
log M, respectively (data not
shown). In contrast, the impaired relaxation responses to isoproterenol
were prevented in RV-exposed tissues that were pretreated with IL-5R
antibody (Fig. 2A) where the mean Rmax and
pD50 values averaged 56.2 ± 5.9% and 6.11 ± 0.11
log M, respectively. Similarly, in comparable experiments, the
attenuated relaxation responses to isoproterenol were also ablated in
RV-exposed ASM that was pretreated with IL-1ra (Fig. 2B). In
contrast to the observations pertaining to RV-exposed ASM, we found
that in control tissues, neither pretreatment with IL-4 NAb, IL-5R
antibody, nor IL-1ra affected the subsequent relaxation responses of
the tissues to isoproterenol (data not shown).
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Regulation of IL-5 expression in RV-inoculated ASM.
The above observations are consistent with those in a recent
study by Hakonarson et al. (17) demonstrating that exposure of isolated
rabbit ASM tissue and cultured rabbit or human ASM cells to RV induces
an upregulated mRNA expression and release of IL-1 protein into the
ASM culture medium, an effect detectable after 6 h of exposure to the
viral pathogen. Moreover, in the latter study, we found that the
RV-induced upregulated release of IL-1
was responsible for the
observed changes in ASM responsiveness in the RV-inoculated ASM (17).
Given these previous findings, when evaluated in light of our above
extended present observations implicating a role for IL-5, the
consideration is raised that IL-5 and IL-1
may share a common or
interactive mechanism in mediating the observed changes in ASM
responsiveness in the RV-exposed state. In addressing this possibility,
a series of experiments was conducted to initially examine whether
cultured human ASM cells are induced to express altered mRNA and
protein release of IL-5 in the RV-exposed state. For analysis of IL-5
mRNA, with the use of RT-PCR and a human IL-5-specific primer, cDNA was
reverse transcribed from total isolated RNA primed with oligo(dT), and Southern blots were subsequently prepared and probed with the human
cDNA probe specific for the human IL-5 gene (see METHODS). In addition, a 157-bp probe for the constitutively expressed RPL7 was
also prepared and used to control for gel loading, and the signals for
the individual PCR products were quantitated with a PhosphorImager. As
shown by one of three representative experiments in Fig.
3, exposure of cultured human ASM cells to
RV induced a marked upregulated expression of the IL-5 mRNA signal,
distinctly evident at 3, 6, and 24 h after RV inoculation. In contrast,
the intensity of the IL-5 mRNA signal obtained in control cells was relatively low and remained essentially unaltered during the 24-h period of exposure to vehicle alone. Of note, the corresponding constitutively expressed RPL7 signal also remained unaltered in both
the control and RV-exposed cells.
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Role of IL-5 in mediating IL-1 release in RV-exposed
ASM.
In considering the above results together with recent findings by
Hakonarson et al. (17) demonstrating that the changes in agonist
responsiveness in RV-exposed ASM are mediated by an induced upregulated
release and autocrine action of IL-1
, we next examined whether the
above observed effects of RV on IL-5 release are mechanistically
coupled to the induction of IL-1
release from RV-exposed ASM cells.
Accordingly, IL-1
protein levels were measured by radioimmunoassay
of the culture medium of human ASM cells exposed for 24 h to vehicle
alone and to RV16 in the absence and presence of IL-4 NAb and IL-5R
antibody. As depicted in Fig. 5, relative
to control cells, the elaboration of IL-1
protein was markedly
increased in RV-exposed ASM cells, and although IL-4 NAb had no effect,
pretreatment of the cells with IL-5R
antibody abrogated their
RV-induced enhanced release of IL-1
. Thus these observations support
the notion that the induced release of IL-5 by RV-inoculated ASM (Fig.
4) was largely responsible for the reported increase in IL-1
release
from RV-exposed ASM (17). Interestingly, Hakonarson et al. (21)
recently demonstrated that a similar sequential pattern of initial IL-5
release that, in turn, mediates a secondary release of IL-1
is
responsible for the induction of comparable changes in agonist
constrictor and relaxant responsiveness in isolated rabbit ASM
passively sensitized with human atopic asthmatic serum.
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DISCUSSION |
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The fundamental mechanism(s) underlying the well-known changes in
airway reactivity that occur after infection of the respiratory tract
with certain viral pathogens remains to be elucidated. In this context,
it is relevant to note that current evidence supports the general
concept that the airway responses in allergic asthma and those obtained
after infection with specific viral respiratory pathogens may share
certain common mechanistic pathways. Accordingly, it is well recognized
that, as in allergic asthma, infection of the respiratory tract with
specific viral pathogens can induce airway inflammation (5, 12),
cytokine release (25, 32, 34, 37), and IgE production (2, 16). Insofar
as these proinflammatory responses may be associated with altered
airway reactivity that is similar to that seen in allergic asthma, it is likely that a mechanistic interplay between specific viral respiratory pathogens and activated inflammatory cells, airway epithelial cells, and other cell types importantly contributes to the
viral-induced pro-asthmatic-like phenotype of altered airway responsiveness. In light of this consideration, together with the
well-documented crucial role of Th2-type cytokines in the pathogenesis
of allergic asthma (1, 11, 24, 29, 36), the present study examined the
potential contributions of the Th2-type cytokines IL-4 and IL-5 in
mediating RV-induced changes in ASM responsiveness. The results
provided new evidence demonstrating that 1) exposure of naive
ASM tissue to RV induced heightened ASM constrictor responsiveness and
attenuated -adrenoceptor-mediated ASM relaxation; 2) these
RV-induced changes in ASM responsiveness were prevented by pretreating
the tissues with either an IL-5R
antibody or human IL-1ra, whereas
an IL-4 NAb had no effect; 3) the RV-induced changes in ASM
responsiveness were associated with an upregulated expression of IL-5
mRNA and increased IL-5 protein release in the RV-exposed state; and
4) the latter enhanced release of IL-5 was largely responsible
for an associated increase in IL-1
release by ASM in the RV-exposed state.
To our knowledge, this study is the first to demonstrate that the
induced endogenous release of IL-5 and IL-1 by RV-exposed ASM
results in its autocrine manifestation of altered agonist responsiveness. In addressing the collection of evidence supporting this central finding, certain issues pertaining to the present observations are worthy of consideration. Among these, it is relevant to note that, in general, our observed changes in constrictor and
relaxant responsiveness in isolated RV-exposed ASM (Figs. 1 and 2)
mimicked the perturbations in airway function that characterize the
asthmatic condition in vivo and isolated asthmatic airways in vitro,
including enhanced bronchoconstrictor responsiveness and impaired
-adrenoceptor-mediated airway relaxation (3, 4, 10, 14, 33).
Moreover, to the extent that these effects were found to be largely
ablated in RV-exposed ASM that was pretreated with IL-5R
antibody or
IL-1ra (Figs. 1 and 2), as a first approximation, these
results suggested that both IL-5 and IL-1
were endogenously released
by the RV-exposed ASM and that these cytokines acted similarly or
interacted cooperatively in ultimately mediating the observed changes
in ASM responsiveness. Both these considerations concur with the
collection of related information based on other published
reports. In this context, previous studies (6, 26, 28, 31)
have demonstrated that apart from certain leukocytes, various non-bone
marrow-derived resident tissue cells (e.g., epithelial cells,
endothelial cells, keratinocytes) can be induced to elaborate specific
cytokines, including Th2-type cytokines. Moreover, Hakonarson and
colleagues (19, 22) recently demonstrated that atopic asthmatic
serum-sensitized ASM cells can also be induced to sequentially release
IL-5 and IL-1
in the atopic sensitized state. Our present observations extend this earlier evidence by demonstrating that ASM
cells can additionally be induced to autologously express IL-5 and
IL-1
and elaborate these cytokines in response to inoculation with
RV (Figs. 4 and 5).
In addressing the potential mechanism by which IL-5 and IL-1 may
interact in mediating the observed changes in ASM responsiveness in the
RV-exposed state, the present study examined the possibility that the
induced release of these cytokines represented a mechanistically related phenomenon. Indeed, as depicted in Fig. 5, when RV-inoculated ASM cells were pretreated with an IL-5-receptor blocking antibody, the
increased elaboration of IL-1
was largely prevented. Thus, when
taken together, the present findings support the notion that our
observed changes in ASM responsiveness in the RV-exposed state largely
reflected the behavior of a cooperative system of sequential autocrine
signaling that involved an initial viral-induced release of IL-5 that,
in turn, elicited the subsequent release of IL-1
by the ASM itself.
Of note, this concept of sequential autocrine signaling involving IL-5
and Il-1
is consistent with the recent findings by Hakonarson et al.
(21) demonstrating a similar pattern of sequential IL-5 and IL-1
release in mediating the changes in agonist responsiveness obtained in
atopic asthmatic-sensitized ASM. Moreover, in this respect, it is
relevant to note that, based on previous findings by Hakonarson and
colleagues (18, 20), the mechanism by which IL-1
release ultimately
leads to changes in ASM responsiveness is apparently due to its
induction of enhanced expression and action of Gi protein,
specifically G
i-2 and G
i-3, which inhibit
intracellular cAMP accumulation.
An important consideration raised by the findings of the present study
relates to the potential relevance of our in vitro observations
to the in vivo condition. In this context, it is generally believed
that the airway responses to respiratory tract infection with RV are
largely mediated by cytokine release from infected and damaged airway
epithelial cells and activated leukocytes infiltrating the affected
airways (25, 32, 34, 37). Our present observations extend this
contemporary view by identifying an important role for RV-induced
cytokine release by the ASM itself in autologously mediating changes in
ASM responsiveness. This evidence concurs with the emerging compelling
concept that the ASM itself constitutes an autologously regulated
system that when activated in the sensitized state (e.g., with RV
infection or with atopic sensitization) elicits the expression of
specific cell surface proteins (e.g., ICAM-1) and the release of
specific cytokines (e.g., IL-1) that lead to pro-asthmatic-like
perturbations in ASM responsiveness (17, 19, 20, 22, 23). Moreover, in
extending this concept, because ICAM-1 activation has been well
established as a crucial mechanism for immune effector cell mobilization and action, our present findings raise the possibility that RV-induced activation of ICAM-1 in ASM may serve to mediate both
the altered ASM responsiveness and the localized accumulation of
inflammatory cells in the airways in the RV-infected state. This
possibility remains to be systematically investigated.
In conclusion, the results of the present study provide new evidence
demonstrating that both the Th2-type cytokine IL-5 and the pleiotropic
proinflammatory cytokine IL-1 are endogenously released by ASM cells
in response to inoculation with RV and that these cytokines act
cooperatively in mediating the induced changes in ASM responsiveness in
the RV-exposed state. In addition, the results demonstrate that the
nature of the latter cytokine interaction is given by IL-5-mediated
induction of the autocrine release and action of IL-1
in the
RV-exposed ASM. Thus together with the conventional concepts related to
the roles of various inflammatory cells in the pathogenesis of the
airway response to RV infection, the present findings identify a
potentially important mechanism by which the resident ASM itself may
autologously regulate its own state of altered reactivity in response
to RV infection. Moreover, in this connection, insofar as Th2-type
cytokines are importantly implicated in the pathobiology of allergic
asthma, the findings of the present study provide new evidence in
support of the notion that the altered airway responses seen in
allergic asthma and in the RV-infected state share a common mechanism
that involves the autocrine release and actions of IL-5 and IL-1
in
the affected ASM.
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ACKNOWLEDGEMENTS |
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We thank J. S. Grunstein for expert technical assistance and M. Brown for typing the manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-31467, HL-58245, and HL-61038.
H. Hakonarson was supported as a Parker B. Francis Fellow in Pulmonary Research.
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, Children's Hospital of Philadelphia, Univ. of Pennsylvania School of Medicine, 34th St. and Civic Center Blvd., Philadelphia, PA 19104 (E-mail: grunstein{at}emailchop.edu).
Received 5 November 1999; accepted in final form 13 January 2000.
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