Divisions of Pulmonary Medicine and Allergy, Immunology, and Infectious Diseases, 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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To elucidate the mechanistic
interplay between rhinovirus (RV) exposure and atopic sensitization in
regulating airway smooth muscle (ASM) responsiveness, isolated rabbit
ASM tissue and cultured human ASM cells were passively sensitized with
sera from atopic asthmatic or nonatopic nonasthmatic (control) subjects
in the absence and presence of inoculation with RV serotype 16. Relative to control subjects, atopic asthmatic serum-sensitized and
RV-inoculated ASM exhibited significantly increased contractility to
acetylcholine, impaired relaxation to isoproterenol, and enhanced
release of the proinflammatory cytokine interleukin-1. These effects
were potentiated in atopic asthmatic serum-sensitized ASM concomitantly inoculated with RV and inhibited by pretreating the tissues with monoclonal blocking antibodies against intercellular adhesion molecule
(ICAM)-1 (CD54), the host receptor for RV serotype 16, or lymphocyte
function-associated antigen (LFA)-1 (CD11a/CD18), the endogenous
counterreceptor for ICAM-1. Moreover, RV inoculation was found to
potentiate the induction of mRNA and surface protein expression of
Fc
RII (CD23), the low-affinity receptor for IgE, in atopic asthmatic
serum-sensitized ASM. Collectively, these observations provide new
evidence demonstrating that 1) RV exposure and atopic
sensitization act cooperatively to potentiate induction of proasthmatic
changes in ASM responsiveness in association with upregulated
proinflammatory cytokine release and Fc
RII expression and
2) the effects of RV exposure and atopic sensitization are mediated by cooperative ICAM-1-coupled LFA-1 signaling in the ASM itself.
asthma; viral infection; cell adhesion molecules; cytokines; Fc receptors
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INTRODUCTION |
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RHINOVIRUS (RV)
infection of the respiratory tract is the most common cause of the
common cold and as such represents the most common infectious disease
in humans. Moreover, relative to other viral respiratory pathogens, RV
infection also represents the most frequent trigger of exacerbation of
asthma symptoms in both asthmatic children and adults (5, 6, 9,
11, 22). In concert with this latter effect, previous studies
(2, 6, 11) have demonstrated that RV infection induces a
transient airway hyperreactivity to bronchoconstrictor stimuli in
asthmatic individuals as well as in normal (i.e., nonasthmatic)
subjects. Although the pathophysiological mechanism(s) underlying
RV-induced exacerbation of asthma symptoms and airway constrictor
hyperreactivity remains to be elucidated, there exists ample evidence
to suggest that the altered airway responsiveness both elicited by RV
infection and phenotypically expressed in atopic asthma share certain
common mechanistic pathways (6, 9, 11, 22). In this
regard, a number of studies (20, 27, 28, 31) conducted on
various cell types (e.g., respiratory epithelium, respiratory
submucosal glands, fibroblasts, monocytes, and macrophages) have
demonstrated that infection with RV elicits the expression and release
of a variety of proinflammatory cytokines including interleukin
(IL)-1, IL-1
, IL-6, IL-8, IL-9, IL-11, tumor necrosis factor-
,
and granulocyte-macrophage colony-stimulating factor. As in allergic
asthma, it is generally believed that the actions of these cytokines
individually or in combination likely underlie the clinical
manifestations of symptomatic RV infection. Moreover, in relating this
concept to the pathogenesis of RV-induced changes in airway
responsiveness, we recently identified that apart from the above cell
types, RV is also capable of directly infecting airway smooth muscle
(ASM) cells and elicits "proasthmatic-like" changes in ASM tissue
responsiveness secondary to induced endogenous release and autocrine
action of the pleiotropic proinflammatory cytokine IL-1
by the
RV-exposed ASM itself (14, 19). To the extent that
these RV-mediated effects on IL-1
release and ASM responsiveness are
qualitatively similar to those obtained in ASM passively sensitized
with human atopic asthmatic serum (14, 16, 17), the
collection of findings supports the existence of an important
mechanistic interplay between RV infection and atopic asthmatic airway
sensitization involving the ASM itself. In addressing this issue, the
present study examined the mechanisms underlying the interactive
effects of RV inoculation and atopic asthmatic serum sensitization of
ASM on agonist-mediated ASM constrictor and relaxant responsiveness.
The results provide new evidence demonstrating that 1) RV
inoculation and atopic sensitization of ASM act cooperatively to
potentiate the induction of proasthmatic-like changes in
proinflammatory cytokine release and ASM responsiveness; 2)
the latter phenomena are associated with complementary RV-induced potentiated upregulation of the expression of the low-affinity receptor
for IgE, Fc
RII, in ASM; and 3) the combined effects of RV
exposure and atopic sensitization are attributed to cooperative signaling induced by coligation of intercellular adhesion molecule (ICAM)-1, the cell surface receptor for most RV subtypes, with its
endogenous
2-integrin counterreceptor ligand lymphocyte
function-associated antigen (LFA)-1 in the ASM itself. Collectively,
these findings identify that the induction of ICAM-1-coupled LFA-1
signaling in ASM represents a cooperative mechanism that underlies the
interaction between atopic sensitization and RV exposure in regulating
the phenotypic expression of proasthmatic changes in airway responsiveness.
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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 the study.
Preparation and sensitization of rabbit ASM tissue. After general anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg), the rabbits were killed with an overdose of pentobarbital sodium (130 mg/kg). As previously described (16, 17), the tracheae were removed via an open thoracotomy, the loose connective tissue and epithelium were scraped and removed, and the tracheae were divided into eight ring segments of 6-8 mm in length. Each alternate ring was incubated for 24 h at room temperature in either 1) human serum containing >1,000 IU/ml of IgE obtained from allergic patients with moderate to severe asthma who demonstrated 4-5 or 6+ radioallergosorbent test (RAST)-positive specific IgE concentrations of >17.5 Phadebas RAST units/ml to Dermatophagoides pteronyssimus, D. farinae, and ragweed and who had positive skin tests to these antigens or 2) human serum from nonatopic nonasthmatic (control) individuals with normal serum IgE levels (i.e., <70 IU/ml) and negative skin test reactivity to D. pteronyssimus, D. farinae, and ragweed. In parallel experiments, ASM segments incubated in control serum and atopic asthmatic serum were inoculated with RV serotype 16 (RV16), prepared as described in Culture of human RV, in the absence and presence of maximum effective concentrations of either an IgG1-type anti-ICAM-1 monoclonal blocking antibody (MAb), an IgG2A-type anti-CD11a MAb, or an IgG1-type anti-CD11b MAb. All the tissues studied were aerated with a continuous supplemental O2 mixture (95% O2-5% CO2) during the incubation phase.
Culture of human RV.
A stock solution of human RV16 was prepared by infecting monolayer
cultures of human embryonic lung fibroblasts (MRC-5) with freshly
isolated RV16 from the Clinical Virology Laboratory (Children's Hospital of Philadelphia). As previously described (14,
19), 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 × 104 virus particles/aliquot) at
70oC for use in the experiments described in
Preparation and RV inoculation of cultured human ASM cells.
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 each tracheal segment 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, 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
(17). 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 acetylcholine (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, expressed as the negative logarithm of the
concentration 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.
Preparation and RV inoculation of cultured human ASM cells.
Cultured human ASM cells were obtained from Clonetics (San Diego, CA).
The ASM cells were derived from two male donors 16 and 21 yr of age who
had no evidence of lung disease. The cells were carefully characterized
by the manufacturer with specific markers to confirm their selective
smooth muscle phenotype and to exclude contamination with other cell
types. The cells 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 (human recombinant), 2 ng/ml of fibroblast
growth factor (human recombinant), 50 ng/ml of gentamicin, and 50 ng/ml of amphotericin B. The experimental protocol involved growing the cells
to confluence in the above medium. Thereafter, the cells were starved
in unsupplemented SMBM for 24 h, at which time the cells were
treated for 0, 3, 6, and 24 h with either human control serum,
human atopic asthmatic serum, or serum-free medium alone, each in the
presence and absence of RV16 as previously described (16,
17). The cells were then examined for elaboration of IL-1
protein and expression of Fc
RII mRNA as described in ELISA measurement of IL-1
protein release and Determination of
Fc
RII mRNA expression in human ASM cells, respectively.
ELISA measurement of IL-1 protein release.
IL-1
protein levels were assayed in the culture medium of ASM
tissues and cells that were exposed for 24 h to either RV16 or
vehicle alone under the above conditions of serum sensitization in both
the absence and presence of anti-CD11a MAb or anti-CD11b MAb. The
IL-1
protein levels were quantitatively assessed with an
enzyme-specific immunoassay as previously described (14, 16). The latter assay was performed with a double-antibody
sandwich strategy in which an acetylcholinesterase-F(ab)-conjugated
IL-1
-specific secondary antibody is targeted first to an
IL-1
-captured antibody. The enzymatic activity of
acetylcholinesterase was measured spectrophototometrically, and
relative to a linear standard curve, the results were used to quantify
the amount of the targeted IL-1
present in the culture medium.
Determination of FcRII mRNA expression in human ASM cells.
Total RNA was isolated from the ASM cell preparations with the modified
guanidinium thiocyanate-phenol-chloroform extraction method that
included proteinase K (in 5% SDS) for digestion of protein in the
initial RNA pellet as previously described by our laboratory
(15). 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 Fc
RII, we used a RT-PCR
protocol that included human-specific primers for the gene as well as
for the constitutively expressed ribosomal protein (RP) L7 gene. cDNA
was synthesized from the total RNA isolated from ASM cells exposed for
0, 3, 6, and 24 h to RV or vehicle alone. The cDNA was primed with
oligo(dT)12-18 and extended with Superscript II
reverse transcriptase (GIBCO BRL). PCR was used to amplify the specific
products from each cDNA reaction based on the published sequences of
the human Fc
RII and RPL7 genes and included the following primer
sets: 5'-CGTCTCTCAAGTTTCCAAG-3' (5' primer) and
5'-GCACTTCCGTTGGAATTTG-3' (3' primer) for Fc
RII (product is
333 bp) and 5'-AAGAGGCTCTCATTTTCCTGGCTG-3' (5' primer) and
5'-TCCGTTCCTCCCCATAATGTTCC-3' (3' primer) for RPL7 (product is 157 bp).
The cycling profile used was denaturation at 95°C for 1 min,
annealing at 52-55°C for 1.0 min, and extension at 72°C for
1.0 min, with 30 and 26 cycles for the Fc
RII and RPL7 genes,
respectively. The number of cycles was determined to be in the linear
range of the PCR products. PCRs for the Fc
RII and RPL7 primers were
performed with equivalent amounts of cDNA prepared from 2.5 µg of
total RNA. Equal aliquot of each PCR was 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 Fc
RII and RPL7 DNA levels were assayed by Southern blot analysis with
32P-labeled probes prepared by pooling several RT-PCRs for
the individual Fc
RII 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: one time for 15 min in 2× saline-sodium
citrate (SSC)-0.1% SDS; one time for 15 min in 0.1× SSC-0.1% SDS
(both at room temperature), and two times for 1 min at 50°C in 0.1× SSC-0.1% SDS.
Determination of FcRII protein expression in ASM tissue.
Expression of cell surface Fc
RII protein was assayed by Western blot
analysis of membrane protein samples isolated from ASM tissues exposed
for 24 h to control or atopic asthmatic serum in both the absence
and presence of RV. The ASM tissues were minced and homogenized in 50 mmol/l of Tris · HCl, 150 mmol/l of NaCl, and 1 mmol/l of EDTA
(pH 7.4) containing 1 mmol/l of phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, and 5 µg/ml of leupeptin. Nuclei and large
particulates were removed by centrifugation at 100 g for 5 min. The supernatant was then centrifuged at 100,000 g for 1 h to pellet the membrane fractions. The membrane pellet was resuspended in the same Tris-EDTA buffer, and the protein concentration was measured. Equivalent amounts (30-50 µg) of membrane protein were fractionated in 11% SDS-polyacrylamide gels followed by transfer to nitrocellulose membranes. The membranes were then blotted overnight at room temperature in 25 mmol/l of Tris · HCl (pH 7.5), 150 mmol/l of NaCl, and 0.05% Tergitol Nonidet P-40 containing 5% nonfat milk as previously described by our laboratory (15). The
primary mouse anti-human Fc
RII antibody used was diluted 1:250 to
1:500 and incubated for 1 h at room temperature. The primary and
secondary antibody incubations and washes were done in 25 mmol/l of
Tris · HCl (pH 7.5), 150 mmol/l of NaCl, and 0.05% Nonidet
P-40 containing 0.50% nonfat milk. The Fc
RII receptor levels were
detected with enhanced chemiluminescence after a 1-h incubation with a
1:1,000 dilution of an anti-mouse horseradish peroxidase-linked
secondary antibody and subsequent exposure to autoradiography film.
Expression levels of the Fc
RII protein were quantitated by laser
densitometry (Bio-Rad, Hercules, CA).
Reagents.
The human ASM cells and SMBM were obtained from Clonetics. The FcRII
and RPL7 primers were obtained from Integrated DNA Technologies (Coralville, IA). The anti-ICAM-1, anti-CD11a, and anti-CD11b neutralizing antibodies; the IL-1
ELISA kit; 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
3 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 or ANOVA with multiple comparison of means, where appropriate. P values < 0.05 were considered significant.
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RESULTS |
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Complementary effects of RV and atopic sensitization on ASM
responsiveness.
Agonist-induced constrictor and relaxation responses were separately
examined in isolated paired rabbit tracheal ASM segments that were
incubated for 24 h in either human atopic asthmatic serum or serum
from nonatopic nonasthmatic (control) individuals in both the absence
and presence of concomitant treatment with RV16. As shown in Fig.
1, relative to control tissues, the
Tmax responses to exogenously administered ACh were
significantly increased in ASM exposed to RV and in tissues passively
sensitized with atopic asthmatic serum (P < 0.01).
Accordingly, the Tmax values amounted to 95.7 ± 11.4, 143.5 ± 12.4, and 159.8 ± 12.5 g/g ASM weight in the
control, RV-exposed, and atopic asthmatic serum-sensitized tissues,
respectively, representing average RV- and atopic asthmatic serum-induced increases in Tmax of ~50 and 67% above
control values, respectively. The corresponding sensitivities to ACh
(pD50; i.e., log ED50 values) amounted to
5.02 ± 0.04, 5.15 ± 0.07, and 5.30 ± 0.06
log M,
respectively, demonstrating that, relative to control values, the
pD50 values were also significantly increased
(P < 0.05) in the atopic asthmatic serum-sensitized
and RV-exposed tissues. Of note, in atopic asthmatic serum-sensitized
tissues concomitantly exposed to RV (Fig. 1), the Tmax and
pD50 values amounted to 152.1 ± 11.9 g/g ASM and
5.16 ± 0.06
log M, respectively, and although both these
determinations were significantly increased relative to the control
value (P < 0.05), the values were similar to those
obtained in tissues exposed to RV or to the atopic-sensitizing serum
alone.
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Role of ICAM-1/LFA-1 signaling in mediating the complementary
effects of RV and atopic sensitization on ASM responsiveness.
Using the same experimental preparation described herein, Grunstein et
al. (12) recently reported that the changes in
constrictor and relaxant responsiveness obtained in atopic asthmatic
serum-sensitized ASM segments were prevented by pretreating the tissues
with either a MAb to ICAM-1 or a MAb directed specifically against the
-chain of the
2-integrin cell adhesion molecule LFA-1
(i.e., anti-CD11a MAb), which serves as an endogenous counterreceptor
ligand for ICAM-1 (12). In light of this evidence,
together with the fact that in addition to its binding to
2-integrins, ICAM-1 also represents the principal host
receptor for most RV subtypes (10, 29), studies were
conducted to determine the potential contribution of ICAM-1/LFA-1
coupling in mediating the combined effects of RV exposure and atopic
asthmatic serum sensitization on ASM agonist responsiveness. As
depicted in Fig. 3, relative to control
serum-exposed tissues, ASM concomitantly sensitized with atopic
asthmatic serum and inoculated with RV depicted significantly enhanced
Tmax responses to ACh (P < 0.01), and
these augmented constrictor responses were largely ablated in atopic
asthmatic serum-sensitized plus RV-exposed tissues that were pretreated
with either anti-ICAM-1 MAb (Fig. 3A) or anti-CD11a MAb
(Fig. 3B). Comparably, relative to control tissues, atopic
asthmatic serum-sensitized plus RV-exposed ASM also displayed
significantly impaired relaxation responses to isoproterenol
(P < 0.01), and this attenuating effect on ASM
relaxation was also completely abrogated in atopic asthmatic
serum-sensitized plus RV-exposed tissues that were pretreated with
either anti-ICAM-1 MAb (Fig.
4A) or anti-CD11a MAb (Fig.
4B). In contrast to these observations, neither anti-ICAM-1
MAb nor anti-CD11a MAb was found to appreciably affect the ASM
constrictor responses to ACh or relaxation responses to isoproterenol
in control tissues (data not shown). Further contrasting the above
results, pretreatment of atopic asthmatic serum-sensitized plus
RV-inoculated ASM with a blocking MAb directed specifically against
another
2-integrin, Mac-1 (i.e., CD11b/CD18), had no
effect on the heightened constrictor responsiveness of the tissues to
ACh or impaired relaxation responsiveness to isoproterenol (data not
shown). Moreover, in extended experiments designed to further
substantiate the specificity of the above protective actions of
anti-ICAM-1 MAb and anti-CD11a MAb in atopic asthmatic serum-sensitized
plus RV-exposed ASM, we found that neither of these MAbs significantly
affected the altered agonist responsiveness elicited in ASM exposed for
24 h to exogenously administered IL-1
(20 ng/ml; data not
shown), a condition previously shown to produce changes in ASM
constrictor and relaxant responsiveness qualitatively similar to those
exhibited above in atopic asthmatic serum-sensitized and RV-inoculated
ASM (14).
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Cooperative effects of RV and atopic sensitization on IL-1
release from ASM.
In separate studies, Hakonarson and colleagues (14, 16,
18) recently reported that RV inoculation and atopic asthmatic serum sensitization of ASM independently induce the release of the
proinflammatory cytokine IL-1
from the ASM itself, an effect that is
responsible for the induced changes in ASM responsiveness under each of
these experimental conditions. Given this evidence, a series of
experiments was conducted to examine the role and mechanism of
interaction between atopic asthmatic serum sensitization and RV
exposure in regulating IL-1
protein release from ASM. IL-1
protein levels were measured by radioimmunoassay of the culture medium
of rabbit ASM tissue preparations and cultured human ASM cells exposed
for 24 h to either control or atopic asthmatic serum in both the
absence and presence of RV. The IL-1
protein levels in the control
serum-treated ASM tissue and cultured cell preparations averaged
6.42 ± 1.04 pg/g ASM and 3.90 ± 0.62 pg/ml, respectively.
As shown in Fig. 5, relative to these
respective control values, the IL-1
protein levels in the culture
medium were significantly increased in both the rabbit ASM tissue (Fig. 5A) and human ASM cell preparations (Fig. 5B)
subjected to either atopic asthmatic serum sensitization or RV exposure
alone. Moreover, it will be noted that the elaboration of IL-1
was
further enhanced in the ASM tissue and cultured ASM cell preparations
that were concomitantly exposed to atopic asthmatic serum and RV.
Accordingly, relative to the independent effects of atopic asthmatic
serum sensitization and RV exposure alone, where the IL-1
levels
averaged 24.12 ± 5.27 and 16.88 ± 6.52 pg/g ASM,
respectively, the augmented release of IL-1
from atopic asthmatic
serum-sensitized plus RV-exposed rabbit ASM tissues appeared roughly
additive in nature, amounting to 37.55 ± 4.84 pg/g ASM (Fig.
5A). Interestingly, combined atopic asthmatic serum
sensitization and RV exposure of the cultured human ASM cells elicited
a proportionately greater (synergistic) elaboration of IL-1
than
could be accounted for by a simple additive effect of each treatment
condition (Fig. 5B). Finally, as further depicted in Fig. 5,
it will be noted that pretreatment with anti-CD11a MAb completely
ablated the heightened release of IL-1
in both the atopic asthmatic
serum-sensitized plus RV-exposed ASM tissue (Fig. 5A) and
cultured ASM cell preparations (Fig. 5B).
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Modulatory effects of RV on FcRII expression.
In light of the above observations, studies were conducted to further
investigate the nature of the cooperative interaction between atopic
asthmatic serum sensitization and RV exposure in ASM. In this context,
given the previous findings by Hakonarson et al. (15) that
the observed effects of atopic asthmatic serum sensitization on ASM
responsiveness are attributed to induced enhanced expression and
activation of the low-affinity receptor for IgE, Fc
RII (i.e., CD23),
in ASM after its exposure to the high IgE-containing sensitizing serum,
we examined whether RV inoculation of cultured human ASM cells induces
changes in their endogenous expression of Fc
RII. Analysis of
Fc
RII mRNA expression was conducted in ASM cells at various times
after exposure of the cells to RV16 or to vehicle alone (control). For
mRNA analysis, Southern blots were prepared and probed with a cDNA
probe specific for human Fc
RII as well as with a 157-bp probe for
the constitutively expressed RPL7 gene (see METHODS). As
illustrated by a representative experiment in Fig.
6, relative to the corresponding
constitutively expressed RPL7 signal, mRNA expression of Fc
RII under
control conditions was only modestly detected at various times after
exposure of the cells to vehicle alone. In contrast, relative to the
unaltered control Fc
RII and corresponding RPL7 signals, the
intensity of the Fc
RII mRNA signal was progressively enhanced at 3, 6, and 24 h after exposure of the cells to RV.
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DISCUSSION |
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Naturally occurring and experimentally induced RV infections of
the respiratory tract have been associated with elicitation of a
transient increase in bronchoconstrictor responsiveness in asthmatic
individuals as well as in normal (i.e., nonasthmatic) subjects
(2, 6, 9, 11, 22). Although the mechanism(s) underlying
the RV-induced changes in airway responsiveness remains to be
identified, it is generally believed that this effect is a consequence
of the inflammatory response to RV infection in the affected airway
that involves the evoked release and actions of a host of cytokines
generated by various cell types including specific leukocytes,
respiratory epithelial cells, and fibroblasts (20, 27, 28,
31). Moreover, in a recent study (19), it has been
demonstrated that RV is also capable of directly infecting ASM cells
and that RV inoculation of ASM tissue elicits proasthmatic-like changes
in the responsiveness of the tissue to airway constrictor and relaxant
agonists. The latter phenomenon was attributed to an induced autocrine
release and action of IL-1 in the RV-exposed ASM itself
(14), and both the induction of IL-1
release and altered ASM responsiveness were completely abrogated in RV-exposed ASM
that was pretreated with a MAb directed against ICAM-1
(14), the host receptor for the vast majority (i.e.,
"major group") of RVs (10, 29). In light of this
evidence, together with the fact that the evoked release of IL-1
and
altered agonist responsiveness in RV-exposed ASM are effects
qualitatively similar to those independently reported in atopic
asthmatic-sensitized ASM (16, 17), the present study
examined the potential mechanistic interplay between RV exposure and
atopic asthmatic airway sensitization in regulating ASM responsiveness.
Collectively, the results provide new evidence demonstrating that
1) RV inoculation and atopic sensitization of ASM act
cooperatively to potentiate the induction of proasthmatic-like changes
in ASM responsiveness and IL-1
release; 2) the latter phenomena are associated with RV-induced upregulation of the expression of the low-affinity receptor for IgE, Fc
RII, in ASM; and
3) the cooperative effects of RV exposure and atopic
sensitization are mediated by induced ICAM-1-coupled LFA-1 signaling in ASM.
To our knowledge, this study is the first to demonstrate that direct RV inoculation and atopic sensitization of ASM elicit complementary changes in ASM responsiveness. In general, this finding supports the clinical observations that imply a synergistic interaction between viral respiratory infections and respiratory allergens in inducing altered airway function and wheezing in asthmatic children and adults (5, 9, 22). In this regard, our observed changes in maximal ASM relaxant responsiveness to isoproterenol (Rmax) demonstrated a near-additive attenuating effect of combined exposure to RV and atopic sensitization (Fig. 2), suggesting the presence of a cooperative interaction between the signaling mechanisms that modulate ASM relaxation in the RV-exposed and atopic-sensitized states. Contrasting these results pertaining to ASM relaxation, although RV exposure and atopic sensitization independently produced significant increases in maximal ASM contractility to ACh (Tmax), the combination of RV inoculation and atopic sensitization failed to produce any further significant changes in Tmax (Fig. 1). Among other possibilities, a reasonable explanation of this finding is that a maximal (or near-maximal) potential increase in Tmax was achieved with either RV exposure or atopic sensitization alone, rendering the ASM incapable of a further increase in contractility in response to both treatment conditions combined.
In addressing the mechanism(s) underlying the apparent cooperative
interaction between RV exposure and AS sensitization in inducing the
observed changes in ASM responsiveness, we examined the potential role
of ICAM-1-coupled engagement of its endogenous 2-integrin counterreceptor ligand LFA-1. Our rationale
for examining this mechanism was based on a collection of earlier
information demonstrating that 1) activation of ICAM-1/LFA-1
coupling is crucial for the induction of airway hyperreactivity in
various animal models of allergic asthma (3, 4, 21, 24,
30); 2) apart from binding to
2-integrins, ICAM-1 also serves as the host receptor for
the major group of RVs (10, 29); 3) the
specific binding sites for LFA-1 and RV on the ICAM-1 molecule are
distinctly different (1, 23, 25, 26); 4) ASM
cells constitutively express both ICAM-1 and LFA-1 on their cell
surface (12); and 5) endogenous ICAM-1/LFA-1
coligation in ASM mediates the altered agonist responsiveness phenotypically expressed in atopic asthmatic serum-sensitized ASM
(12). In concert with this collection of earlier evidence, our results herein demonstrated that the combined effects of RV inoculation and atopic sensitization on ASM responsiveness were inhibited in the presence of either an anti-ICAM-1 or anti-CD11a MAb
(Figs. 3 and 4), whereas an anti-Mac-1-specific MAb had no effect. Thus
these findings are consistent with the notion that the cooperative
effects of RV inoculation and atopic sensitization on ASM
responsiveness are mutually inclusive events mechanistically regulated
by ICAM-1-coupled LFA-1 signaling in ASM.
In extended support of the above concept, our observations demonstrated
that the effects of RV exposure and atopic sensitization on IL-1
release were also mutually inclusive events mediated by ICAM-1-coupled
LFA-1 signaling (Fig. 5). In this regard, it is important to note that,
in separate previous studies (14, 16, 18), the independent
effects of RV inoculation and atopic sensitization on ASM
responsiveness were found to be attributed to the stimulated release
and autocrine action of IL-1
in the ASM itself. Under these
circumstances, the action of IL-1
was found to be largely mediated
by its induced enhanced expression and activation of Gi
protein (specifically, G
i-2 and G
i-3)
that, by inhibiting cAMP accumulation, produced the observed
proasthmatic-like changes in ASM responsiveness (14, 17).
In light of this evidence, our observation of a near-additive combined
effect of RV exposure and atopic sensitization on IL-1
release from
rabbit ASM tissue (Fig. 5A) parallels the observed
near-additive combined effect of these treatment conditions on rabbit
ASM relaxant responsiveness (Fig. 2). Interestingly, in contrast to
rabbit ASM, our results demonstrated a proportionally greater than
additive increase in IL-1
release from cultured human ASM cells
exposed to the combination of RV and atopic asthmatic serum (Fig.
5B). Given this finding, it would be of interest to
determine whether RV inoculation and atopic sensitization exert a
comparable combined synergistic effect on agonist responsiveness in
human ASM tissue. To date, substantial circumstantial evidence based on
clinical studies supports the possibility of such a synergistic
interaction between viral respiratory infections and atopy in inducing
changes in airway function and wheezing in asthmatic individuals
(6, 9, 11, 22).
A fundamental issue raised by the observations of the present study
relates to the mechanism by which RV inoculation and atopic asthmatic
serum sensitization of ASM act cooperatively to elicit ICAM-1-coupled
LFA-1 signaling. In considering this issue, it is relevant to note that
ICAM-1 serves a duality of receptor functions, acting both as the host
receptor for most RV subtypes and as the intrinsic counterreceptor
ligand for LFA-1. Our present observations provide evidence (albeit
indirect) that these dual functions of ICAM-1 are likely manifested in
a mutually inclusive manner in RV-exposed and atopic sensitized ASM. In
this respect, it has been recently established that the binding sites
for both the major group of RVs and LFA-1 are situated on the
extracellular IgD1 domain of the ICAM-1 molecule and, although somewhat
overlapping, these binding sites are distinctly different (1, 23,
25, 26). Thus it is conceivable that RV binding to ICAM-1 may
not interfere with engagement of ICAM-1 and LFA-1. Indeed, given our present findings, the intriguing possibility is raised that RV binding
to ICAM-1 may actually serve to facilitate endogenous ICAM-1-coupled
LFA-1 ligation. Moreover, within the extended context of the present
observations, given the established role of FcRII activation in
mediating our observed effects of atopic asthmatic serum sensitization
on ASM responsiveness (15), the consideration is raised
that activation of ICAM-1/LFA-1 coupling in atopic asthmatic serum-sensitized ASM represents a transmembrane signaling event that is
triggered by Fc
RII activation in the sensitized ASM. Support for the
latter speculated mechanism of action is, in part, provided by recent
reports demonstrating that Fc
RII activation induces enhanced
LFA-1-mediated adhesiveness in CD4+ T cells and increased
cell adhesion molecule expression (7) as well as a
Th2-type profile of cytokine release (8). Thus, in light
of the above considerations, it is reasonable to propose that our
observed combined effect of RV inoculation and atopic sensitization on
ASM responsiveness represents a cooperative interaction between the
above respective mechanisms of ICAM-1/LFA-1 coligation evoked by RV
exposure and atopic sensitization. Clearly, this provocative overall
speculated mechanism of interaction between RV exposure and atopic
sensitization in inducing ICAM-1/LFA-1 coupling in ASM remains to be
systematically investigated in future studies.
In further examining the potential consequences of the interaction
between RV exposure and atopic sensitization in ASM, our extended
studies demonstrated that RV inoculation of cultured human ASM cells
and rabbit ASM tissue elicited an upregulated expression of FcRII
mRNA (Fig. 6) and Fc
RII surface protein (Fig. 7). In considering the
implication of this finding, it is relevant to note that under similar
experimental conditions of atopic asthmatic serum sensitization of
rabbit ASM, Hakonarson et al. (15) previously identified
that Fc
RII expression in ASM is upregulated in the atopic sensitized
state and that this effect is attributed to activation of the
endogenously expressed Fc
RII receptor itself by the elevated IgE
present in the atopic sensitizing serum. Given these earlier
observations, together with the finding that activation of Fc
RII
represents the initial key signaling event that ultimately leads to
induction of the observed changes in ASM responsiveness in the atopic
asthmatic serum-sensitized state (15), the present results
raise the compelling concept that by eliciting an enhanced expression
of Fc
RII, RV inoculation of ASM may render the tissue more
susceptible to the sensitizing effect of the IgE present in the atopic
serum and hence more responsive to inducible changes in constrictor and relaxant responsiveness in the atopic sensitized state. This concept is
supported, at least in part, by the present finding that inoculation of
atopic serum-sensitized ASM with RV induced an increase in Fc
RII
protein expression beyond that elicited by exposure of the tissue to
the atopic sensitizing serum alone (Fig. 7). Insofar as Fc
RII is
also intrinsically expressed in human ASM tissue and its expression is
reportedly increased in ASM isolated from atopic asthmatic patients
(13), it would be important to determine whether the
latter mechanism of facilitated modulation of atopic sensitization by
RV pertains to RV-infected atopic asthmatic individuals.
In conclusion, the present study examined the interactive roles of RV
exposure and atopic sensitization in eliciting changes in ASM agonist
responsiveness. The results provide new evidence demonstrating that RV
inoculation and atopic sensitization of ASM act cooperatively to
potentiate the induction of proasthmatic-like phenotypic changes in ASM
constrictor and relaxant responsiveness and IL-1 release and that
these effects are mediated by induced ICAM-1-coupled LFA-1 signaling in
the ASM. Moreover, the results demonstrate that RV inoculation elicits
an upregulated expression of Fc
RII in ASM, supporting the existence
of a complementary process by which RV exposure and atopic
sensitization mechanistically interact in ASM to facilitate the
expression of proasthmatic changes in airway responsiveness. Thus, in
concert with the conventional concepts related to the interactive roles
of specific cell adhesion molecules and inflammatory cells in the
overall pathobiology of allergic asthma and its exacerbation in
response to RV infection, the present study identifies a potentially
important mechanism by which the resident ASM itself may, via
cooperative intrinsic ICAM-1/LFA-1 signaling, serve to regulate its own
state of altered responsiveness after RV exposure in the atopic
asthmatic condition.
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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 |
---|
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-31467, HL-58245, HL-61038, and HL-59906.
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 (E-mail: grunstein {at}emailchop.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 June 2000; accepted in final form 1 August 2000.
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