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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Cell adhesion
molecules (CAMs) have been importantly implicated in the
pathobiology of the airway responses in allergic asthma, including
inflammatory cell recruitment into the lungs and altered bronchial
responsiveness. To elucidate the mechanism of CAM-related mediation of
altered airway responsiveness in the atopic asthmatic state, the
expressions and actions of intercellular adhesion molecule-1 (ICAM-1)
and its counterreceptor ligand lymphocyte function-associated antigen-1
(LFA-1; i.e., CD11a/CD18) were examined in isolated rabbit airway
smooth muscle (ASM) tissues and cultured human ASM cells passively
sensitized with sera from atopic asthmatic patients or nonatopic
nonasthmatic (control) subjects. Relative to control tissues, the
atopic asthmatic sensitized ASM exhibited significantly enhanced
maximal contractility to acetylcholine and attenuated relaxation
responses to isoproterenol. These proasthmatic changes in agonist
responsiveness were ablated by pretreating the atopic sensitized
tissues with a monoclonal blocking antibody (MAb) to either ICAM-1 or
CD11a, whereas a MAb directed against the related 2-integrin Mac-1 had no effect. Moreover, relative to
control tissues, atopic asthmatic sensitized ASM cells displayed an
autologously upregulated mRNA and cell surface expression of ICAM-1,
whereas constitutive expression of CD11a was unaltered. Extended
studies further demonstrated that 1) the enhanced expression
and release of soluble ICAM-1 by atopic sensitized ASM cells was
prevented when cells were pretreated with an interleukin
(IL)-5-receptor-
blocking antibody and 2) administration of
exogenous IL-5 to naive (nonsensitized) ASM cells induced a pronounced
soluble ICAM-1 release from the cells. Collectively, these observations
provide new evidence demonstrating that activation of the CAM
counterreceptor ligands ICAM-1 and LFA-1, both of which are
endogenously expressed in ASM cells, elicits autologously upregulated
IL-5 release and associated changes in ICAM-1 expression and agonist
responsiveness in atopic asthmatic sensitized ASM.
asthma; interleukin-5; cholinergic contraction; -adrenergic
stimulation; intercellular adhesion molecule-1; lymphocyte
function-associated antigen-1
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INTRODUCTION |
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THE CHARACTERISTIC FEATURES of the airways in bronchial
asthma include enhanced agonist-mediated bronchoconstriction, impaired -adrenoceptor-mediated airway relaxation, and bronchial
inflammation, the latter primarily involving infiltration of the
airways with eosinophils, lymphocytes, and mast cells. In concert with
the contemporary overall view of the development of bronchial
inflammation and its association with changes in airway responsiveness,
a number of studies (6, 13, 32, 39, 43) have identified important roles
for various cell adhesion molecules (CAMs) in orchestrating the net
process of inflammatory cell activation and recruitment into the
affected airways. In this regard, among other relevant cell adhesion
interactions, a critical role for the binding of intercellular adhesion
molecule (ICAM)-1 (CD54) to its
2-integrin counterreceptor ligand lymphocyte function-associated antigen-1 (LFA-1;
i.e., CD11a/CD18) has been demonstrated in a wide variety of cellular
interactions including T-lymphocyte antigen-specific responses,
leukocyte binding to vascular endothelium, and emigration of leukocytes
into inflammatory foci (1, 8, 36). Moreover, in relation to allergic
asthma, ICAM-1 activation has been proposed as mediating the airway
eosinophilia and hyperresponsiveness found in a primate model of
allergic asthma after chronic in vivo antigen challenge (43).
Similarly, a crucial role for ICAM-1 activation in mediating
antigen-induced airway responses was also demonstrated in studies with
other experimental models (4, 5, 26, 33, 37, 44). The collection of
evidence from these studies implicates ICAM-1 activation that is
coupled to leukocyte (principally, eosinophil) influx in mediating the
observed changes in airway responsiveness. However, the mechanistic
interplay between ICAM-1 activation, airway leukocyte infiltration, and
altered airway responsiveness remains largely unidentified. In this
connection, in a recent study (38) conducted on antigen-sensitized
Brown-Norway rats, administration of an anti-ICAM-1 antibody was found
to attenuate the animals' airway constrictor hyperresponsiveness
without producing a concomitant decrease in airway inflammation. Thus
although ICAM-1 activation may importantly contribute to the
development of airway hyperresponsiveness, the mechanism underlying
this phenomenon may not be completely dependent on inflammatory cell
infiltration of the airways. Indeed, in considering the mechanism of
action of ICAM-1, it is relevant to note that, apart from leukocytes per se, cell surface expression of ICAM-1 has also been identified in a
variety of non-bone marrow-derived cell types including certain airway
structural cells and smooth muscle cells (21, 27, 34, 42). Moreover,
expression of ICAM-1 on lung stromal cells and epithelial cells was
found to be upregulated after exposure of asthmatic subjects to
allergen (15, 29), and ICAM-1 expression was also reportedly increased
in bronchial microvascular endothelial cells isolated from asthmatic
individuals (3). Taken together, these findings raise the notion that
ICAM-1 activation on resident airway tissue cells may, at least in
part, contribute to the changes in airway responsiveness that
characterize the proasthmatic phenotype.
In light of the above considerations, together with more recent evidence demonstrating that the airway smooth muscle (ASM) itself can be induced to autologously express and respond to its autocrine release of certain bronchoactive cytokines in the atopic asthmatic sensitized state (17, 19, 20), the present study examined whether ASM endogenously expresses the CAM counterreceptor ligands ICAM-1 and LFA-1 and whether altered ICAM-1/LFA-1 expression and activation leads to cytokine-coupled phenotypic changes in ASM responsiveness in the atopic asthmatic sensitized state. The results provide new evidence demonstrating that 1) pro-asthmatic-like changes in ASM responsiveness obtained in atopic asthmatic serum-sensitized ASM are mediated by ICAM-1/LFA-1 activation; 2) this process is associated with an autologously upregulated cell surface expression of ICAM-1 by the atopic sensitized ASM, whereas LFA-1 expression is unaltered; and 3) the enhanced expression of ICAM-1 is attributed to an induced autocrine release and action of the T helper cell type 2 (Th2)-type cytokine interleukin (IL)-5 in the sensitized ASM itself. Collectively, these findings support the novel concept that activation of the CAM counterreceptor ligands ICAM-1 and LFA-1, both of which are endogenously expressed in ASM cells, leads to cytokine-mediated autocrine changes in ASM responsiveness in the atopic asthmatic sensitized state.
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METHODS |
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Animals. Twenty-two 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.
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 (18), 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 6-8 mm long. 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 concentration of >17.5 Phadebas RAST units/ml) to Dermatophagoides pteronyssimus, D. farinae, and ragweed and who had a positive skin test 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 were incubated in either control serum or atopic asthmatic serum 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.
Preparation 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 any 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, serum-free medium (SFM) alone, or SFM in the presence
of a maximum effective concentration of anti-ICAM-1 MAb, anti-CD11a
MAb, an IL-5-receptor- (IL-5R
) blocking antibody, or
exogenously administered IL-5. The cells were then examined for mRNA
and protein expression of ICAM-1 and CD11a as described in
Determination of ICAM-1 and CD11a mRNA expression in human ASM
cells and Determination of ICAM-1 and CD11a protein expression
in ASM cells by flow cytometry.
Pharmacodynamic studies.
After incubation of the rabbit ASM tissue preparations in control and
atopic asthmatic sera, each 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 with 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 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 (Lo) as previously described by our
laboratory (18). 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 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 the relaxing agent producing 50% of Rmax
(pD50; i.e., geometric mean ED50).
Determination of ICAM-1 and CD11a 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 (16, 17). 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 ICAM-1 and CD11a, we used a RT-PCR protocol that included human-specific primers for these genes as well as for the constitutively expressed ribosomal protein (RP) L7. cDNA was synthesized from total RNA isolated from ASM cells passively sensitized with human control or atopic asthmatic serum. The cDNA was primed with oligo(dT)12-18 and extended with Superscript II RT (GIBCO BRL, Life Technologies). PCR was used to amplify the specific products from each cDNA reaction based on the published sequences of the human ICAM-1, CD11a, and RPL7 genes and including the following primer sets: 5'-GTCCTCTGCTGAGCTTTACA-3' (5'-primer) and 5'-ATCCTTCATCCTTCCAGCAC-3' (3'-primer ) for CD11a (product is 337 bp); 5'-GAGCTGTTTGAGAACACCTC-3' (5'-primer) and 5'-TCACACTTCACTGTCACCTC-3' (3'-primer) for ICAM-1 (product is 367 bp); and 5'-AAGAGGCTCTCATTTTCCTGGCTG-3' (5'-primer) and 5'-TCCGTTCCTCCCCATAATGTTCC-3' (3'-primer) for RPL7 (product is 157 bp).
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, with 35 cycles for ICAM-1 and CD11a and 26 cycles for the RPL7 genes. The number of cycles was determined to be in the linear range of the PCR products. The PCRs for the human ICAM-1, CD11a, 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, 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 ICAM-1, CD11, and RPL7 DNA levels were assayed by Southern blot analysis with 32P-labeled probes prepared by pooling several RT-PCRs for the individual CAM 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 × 1 min at 50°C in 0.1× SSC-0.1% SDS. Southern blots were quantitated by direct measurements of radioactivity in each band with a PhosphorImager (Molecular Dynamics).Determination of ICAM-1 and CD11a protein expression in ASM cells by flow cytometry. Cell surface protein expression of ICAM-1 and CD11a was examined in the cultured human ASM cells with a Coulter EPICS Elite flow cytometer (Coulter EPICS Division, Hialeah, FL) equipped with a 5-W argon laser operated at 488 nM and 300-mW output. Fluorescence signals were accumulated as two-parameter fluorescence histograms, with both percent positive cells and mean channel fluorescence intensity (MFI) being recorded. Cells treated for 24 h with 10% control or 10% atopic asthmatic serum were carefully washed, scraped from the culture flasks, and then resuspended in PBS buffer. The cells were then dispersed by pipetting through a 23-gauge needle and orbital shaking and then stained with mouse anti-human monoclonal antibodies to ICAM-1 and CD11a. To examine for nonspecific binding, the primary antibody was replaced by immunoglobulins of the same isotype following the manufacturer's protocol, with mouse IgG1 as a negative control. After serial washing, the cells were stained with FITC-conjugated goat anti-mouse secondary antibody. The antibody-stained cells were then evaluated by flow cytometry and analyzed with the Elite Immuno 4 statistical software (Coulter EPICS Division). Fluorescence intensities are expressed as percent positive cells as well as MFI.
ELISA measurements of IL-5 and sICAM-1 protein release.
IL-5 and sICAM-1 protein levels were initially measured in the control
and atopic asthmatic sera. Thereafter, the protein levels were assayed
in the culture medium of ASM cells that were exposed for 24 h to either
control or atopic asthmatic serum in both the absence and presence of
specific MAbs, including anti-ICAM-1 MAb, anti-CD11a MAb, or IL-5R
antibody. sICAM-1 protein levels were also assayed in the culture
medium of ASM cells at 0, 3, 6, 12, and 24 h after treatment with SFM
alone or with SFM containing a maximal effective concentration of
IL-5. The IL-5 and sICAM-1 protein levels were quantitatively
assessed with an enzyme-specific immunoassay as previously described by
our laboratory (20). The latter assay was performed with a
double-antibody sandwich strategy in which an
acetylcholinesterase-Fab-conjugated IL-5- or sICAM-1-specific secondary
antibody is first targeted to an IL-5- or sICAM-1-captured antibody.
The enzymatic activity of acetylcholinesterase was measured
spectrophotometrically, and relative to a linear standard curve, the
results were used to quantify the amounts of targeted IL-5 and sICAM-1
present in the cell culture medium.
Reagents.
The human ASM cells and SMBM were obtained from Clonetics (San Diego,
CA). The ICAM-1, CD11a, and RPL7 primers were obtained from Integrated
DNA Technologies, (Coralville, IA). Anti-ICAM-1 MAb, anti-CD11a MAb,
anti-CD11b MAb, anti-IL-5R antibody, the IL-5 and sICAM-1 ELISA
kits, the mouse anti-human ICAM-1 and CD11a primary antibodies, and the
anti-mouse secondary antibody used in the protein assay studies were
purchased from R&D Systems (Minneapolis, MN). The FITC-mouse antibodies
to human ICAM-1 and CD11a used in the flow cytometric studies were
purchased from Caltag. 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 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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Role of ICAM-1/LFA-1 activation in regulating ASM responsiveness in
the atopic asthmatic sensitized state. Agonist constrictor and
relaxation responses were separately examined in isolated rabbit
tracheal ASM segments that were incubated for 24 h in either human
atopic asthmatic serum or serum from nonatopic nonasthmatic (i.e.,
control) individuals in both the absence and presence of an anti-ICAM-1
MAb or blocking antibodies to the natural counterreceptor ligands for
ICAM-1, including LFA-1 (i.e., anti-CD11a MAb) and Mac-1 (i.e.,
anti-CD11b MAb; see METHODS). As depicted in Fig. 1A, relative to control
serum-exposed tissues, the maximal constrictor responses
(Tmax) and sensitivities (pD50; i.e.,
log ED50 values) to exogenously administered ACh
were significantly enhanced in ASM passively sensitized with atopic
asthmatic serum. Accordingly, the mean Tmax values amounted
to 110.0 ± 13.4 and 138.7 ± 15.1 g/g ASM weight in the control and
atopic sensitized tissues, respectively (P < 0.01), and the
corresponding mean pD50 values amounted to 5.28 ± 0.05 and 5.74 ± 0.07
log M, respectively (P < 0.05).
These induced, heightened constrictor responses to ACh were abrogated in atopic sensitized ASM that was pretreated with a maximally effective
concentration (i.e., 350 ng/ml) of anti-ICAM-1 MAb (Fig. 1A).
In these tissues, the mean Tmax and pD50 values
amounted to 108.3 ± 15.0 g/g ASM and 5.22 ± 0.06
log M,
respectively, and the latter determinations were similar to those
obtained in control serum-treated ASM. Similarly, in comparable
experiments, we found that pretreatment of atopic asthmatic
serum-sensitized ASM with anti-CD11a MAb (12.5 µg/ml) also ablated
the heightened constrictor responsiveness of the tissues to ACh (Fig.
1B). Accordingly, in these studies, the Tmax values
obtained in the atopic asthmatic serum-sensitized and control
serum-treated tissues averaged 129.5 ± 7.9 and 104.1 ± 13.2 g/g ASM, respectively (P < 0.01), and the corresponding
pD50 values amounted to 5.03 ± 0.05 and 4.82 ± 0.06
log M, respectively (P < 0.01). In the presence of
anti-CD11a MAb, the Tmax and pD50 values in the
sensitized ASM were similar to those obtained in the control
serum-treated tissues and averaged 101.9 ± 11.2 g/g ASM and 4.79 ± 0.06
log M, respectively. In contrast to these observations, in
separate studies, we found that the augmented constrictor responses to
ACh in atopic sensitized ASM were unaffected by pretreatment of the
sensitized tissues with the anti-Mac-1-specific antibody (anti-CD11b
MAb). In these studies, the mean Tmax and pD50
values averaged 109 ± 8.9 g/g ASM and 5.01 ± 0.05
log M,
respectively, and both values were not significantly different from the
corresponding measurements obtained in sensitized ASM in the absence of
anti-CD11b MAb, which amounted to 113 ± 10.9 g/g ASM and 5.04 ± 0.06
log M, respectively. Moreover, in parallel experiments,
neither anti-ICAM-1 MAb, anti-CD11a MAb, nor anti-CD11b MAb was found
to appreciably affect the ASM constrictor responsiveness to ACh in
control serum-exposed tissues (data not shown).
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In further studies during comparable levels of initial sustained
ACh-induced contractions in control and atopic asthmatic sensitized ASM segments, averaging ~ 50% 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 serum-treated ASM, the Rmax and pD50
values to isoproterenol were significantly attenuated in the
corresponding atopic asthmatic sensitized tissues. Accordingly, the
mean Rmax value for isoproterenol amounted to 22.2 ± 6.1% (SE) in the atopic sensitized ASM compared with 47.7 ± 4.8% in the control serum-exposed ASM (P < 0.005),
and the corresponding pD50 values averaged 6.15 ± 0.05 and 6.36 ± 0.04
log M, respectively (P < 0.05).
These attenuated relaxation responses to isoproterenol were unaffected
in sensitized ASM segments pretreated with anti-CD11b MAb where their
Rmax and pD50 values averaged 27.3 ± 6.1%
and 6.07 ± 0.07
log M, respectively. In contrast, the impaired
relaxation responses to isoproterenol were prevented in atopic
asthmatic sensitized tissues that were pretreated with anti-ICAM-1 MAb
(Fig. 2A) where the mean Rmax and pD50
values averaged 45.2 ± 4.9% and 6.35 ± 0.05
log M,
respectively. Similarly, in comparable experiments, the impaired
isoproterenol-mediated relaxation responses were also ablated in atopic
sensitized tissues that were pretreated with anti-CD11a MAb (Fig.
2B). In contrast to these findings obtained in atopic asthmatic
serum-sensitized ASM, in tissues incubated with control serum,
pretreatment with either anti-ICAM-1 MAb, anti-CD11a MAb, or anti-CD11b
MAb had no effect on the subsequent relaxation responsiveness of the
tissues to isoproterenol (data not shown).
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Expression of ICAM-1 and LFA-1 in atopic asthmatic serum-sensitized
ASM cells.
Taken together, the above observations suggested a role for induced
ICAM-1/LFA-1 activation in mediating the observed changes in ASM
responsiveness in the atopic asthmatic sensitized state. To elucidate
whether this role of ICAM-1/LFA-1 activation represents a phenomenon
that is intrinsic to the atopic sensitized ASM itself, a series of
experiments were conducted to examine whether cultured human ASM cells
autologously express the mRNAs and proteins for the above cell adhesion
molecules and to assess whether the expression of ICAM-1 and LFA-1 in
ASM cells is altered in the atopic asthmatic sensitized state. For the
mRNA analyses, Southern blots were prepared and probed with the human
cDNA probes specific for the human CD11a and ICAM-1 genes (see
METHODS). In addition, a 157-bp RPL7 probe was also used as
a control for gel loading. As depicted by one of three representative
experiments in Fig. 3, relative to the corresponding constitutively expressed RPL7 signal, mRNA expression of
CD11a was modestly detected at various times after exposure of the
cultured ASM cells to control or atopic asthmatic serum. Moreover,
there were no pronounced differences in the intensities of the CD11a
mRNA signals between the control and atopic asthmatic serum-sensitized
cells. In contrast to these observations, as further shown in Fig. 3,
relative to control serum-treated ASM cells where ICAM-1 mRNA
expression was barely detectable after 3, 6, and 24 h of serum
exposure, the intensity of the ICAM-1 mRNA signal was markedly
upregulated at all these times after exposure of the ASM cells to
atopic asthmatic sensitized serum.
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Role of IL-5 in regulating ICAM-1 expression in atopic asthmatic
serum-sensitized ASM cells.
Previous studies (23, 28, 32, 39) have demonstrated increased ICAM-1
expression and release of sICAM-1 into the lungs of asthmatic
individuals and in animal models of allergic asthma. Moreover, in a
recent study (35), inhalation of recombinant human IL-5 was found to
elicit increases in the sputum concentration of sICAM-1 in
atopic asthmatic subjects. This evidence, when coupled to recent
observations by Hakonarson and colleagues (19, 20) demonstrating that human ASM cells are autologously induced to release
IL-5 protein in the atopic asthmatic sensitized state raises the
consideration that IL-5 may play a role in regulating the above
observed changes in ICAM-1 expression in the atopic asthmatic
serum-sensitized ASM cells. In addressing this possibility, studies
were conducted to initially examine whether the reported elaboration of
IL-5 protein by atopic asthmatic serum-sensitized human ASM cells is
mechanistically coupled to ICAM-1/LFA-1 activation. With the use of an
IL-5-specific immunoassay (see METHODS), IL-5 protein
levels were measured in the culture medium of human ASM cells after 24 h of exposure of the cells to control serum or to atopic asthmatic
serum in the absence and presence of anti-ICAM-1 MAb and anti-CD11a
MAb. As shown in Fig. 5, relative to
control serum-treated cells, IL-5 protein levels in the culture medium of atopic serum-sensitized cells were significantly increased by an
average of approximately fourfold. Moreover, it will be noted that this
enhanced elaboration of IL-5 protein was ablated in atopic asthmatic
serum-sensitized ASM cells that were pretreated with either anti-ICAM-1
MAb or anti-CD11a MAb (Fig. 5).
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DISCUSSION |
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Substantial evidence accumulated in recent years has implicated a crucial role for CAMs in the overall pathobiology of the airway responses in allergic asthma, including the development of airway inflammation and the associated changes in airway responsiveness. In this regard, a number of studies (4, 5, 24, 26, 33, 38, 43, 44) have demonstrated that administration of blocking antibodies to specific CAMs including ICAM-1, LFA-1, and very late activating antigen-4 attenuates or abolishes the airway constrictor hyperresponsiveness and/or airway inflammation seen in different in vivo animal experimental models of asthma. For the most part, these studies have largely suggested that the specific CAM-dependent changes in airway responsiveness and airway inflammatory cell infiltration are casually related. More recent reports (24, 26, 31, 38), however, have provided compelling evidence that certain CAM-dependent effects on airway responsiveness may occur independent of leukocyte migration into the lungs. In light of this emerging new information, the consideration is raised that the reported protective effects of specific anti-CAM antibodies on airway hyperresponsiveness may be attributed to a more direct action(s) of CAM activation on resident airway tissue cells. In addressing this issue, the present study examined the role and mechanism of action of ICAM-1-coupled LFA-1 activation in regulating agonist responsiveness in atopic asthmatic serum-sensitized isolated ASM. Collectively, the results herein provide new evidence demonstrating that 1) the proasthmatic perturbations in ASM responsiveness seen in atopic asthmatic sensitized tissues are mediated by ICAM-1/LFA-1 activation; 2) atopic asthmatic serum-sensitized ASM cells also display an autologously upregulated mRNA and protein expression of ICAM-1, whereas CD11a expression is constitutively present on the surface of ASM cells but is unaltered in the sensitized state; and 3) the enhanced elaboration of sICAM-1 by atopic sensitized ASM cells is attributed to an ICAM-1/LFA-1-dependent autocrine release and action of the Th2-type cytokine IL-5 in the ASM itself.
To our knowledge, this study is the first to demonstrate that both
ICAM-1 and its endogenous 2-integrin counterreceptor
CD11a are constitutively expressed in ASM and that activation of these CAM counterreceptor ligands in atopic asthmatic sensitized ASM mediates
its proasthmatic phenotype of altered agonist responsiveness. In
evaluating the collection of evidence supporting these central findings, certain issues pertaining to the present observations are
worthy of consideration. Among these, it is relevant to note that our
observed changes in constrictor and relaxant agonist responsiveness in
atopic asthmatic serum-sensitized ASM tissues (Figs. 1 and 2) mimicked
the perturbations in airway function that characterize the in vivo
asthmatic condition, including augmented bronchoconstrictor
responsiveness and impaired
-adrenoceptor-mediated airway relaxation
(2, 14, 18). Moreover, to the extent that our observed changes in ASM
responsiveness were ablated in atopic sensitized tissues that were
pretreated with blocking antibodies to either ICAM-1 or CD11a, these
results suggested that both ICAM-1 and LFA-1 were expressed by ASM
cells and that activation of these CAM counterreceptors in the atopic
sensitized state mediated the observed changes in ASM responsiveness.
In general, these considerations are consistent with a collection of
related information based on other published reports. In this regard,
previous studies (21, 27, 34, 42) have demonstrated that, apart from
leukocytes, ICAM-1 is also expressed in various non-bone marrow-derived
resident tissue cells (e.g., epithelial cells, endothelial cells)
including ASM cells. Furthermore, as noted above, a host of studies (4, 5, 26, 33, 38, 43, 44) have demonstrated that activation of ICAM-1 and
LFA-1 is crucial for the induction of airway hyperreactivity in various
animal models of allergic asthma. Our present findings extend this
earlier evidence by demonstrating that both ICAM-1 and LFA-1 are
constitutively expressed in ASM cells (Fig. 4) and that their
activation in the atopic sensitized state leads to altered ASM
responsiveness in association with an induction of IL-5 release (Fig.
5) and enhanced ICAM-1 expression and ICAM-1 protein release
(Fig. 6).
Our rationale for investigating the role of IL-5 in atopic asthmatic sensitized ASM is based on considerable evidence implicating an important contribution of this Th2-type cytokine in regulating the induction of airway constrictor hyperresponsiveness in various in vivo animal models of allergic asthma (7, 9, 22, 41). Moreover, in this connection, Hakonarson and colleagues (19, 20) have recently demonstrated that independent of the presence of inflammatory cells, IL-5 is endogenously released by atopic asthmatic serum-sensitized ASM cells and serves to mediate the altered constrictor and relaxant responsiveness in isolated sensitized ASM tissue. In extending these recent findings, our present observations demonstrated that the induced release of IL-5 by atopic asthmatic sensitized ASM cells was inhibited in the presence of either anti-ICAM-1 MAb or anti-CD11a MAb (Fig. 5), implicating ICAM-1/LFA-1 activation in the induction of IL-5 release in the sensitized state. These observations fundamentally concur with those of previous studies (12, 25, 30, 40) that also demonstrated that ICAM-1/LFA-1 activation elicits the release of proinflammatory cytokines (including IL-5) in other cell types.
Although the previous findings by Hakonarson et al. (19) demonstrated that the autocrine release and action of IL-5 in ASM mediate its altered responsiveness in the atopic asthmatic sensitized state, the present results provide additional evidence that ICAM-1/LFA-1-dependent IL-5 release is also responsible for inducing an enhanced elaboration of sICAM-1 from atopic asthmatic serum-sensitized ASM cells (Fig. 6). This finding is in agreement with those of a recent study (35) demonstrating that inhalation of recombinant human IL-5 produces increases in the sputum concentration of sICAM-1 in atopic asthmatic individuals. Moreover, in the latter study, because the sputum concentrations of sICAM-1 obtained after IL-5 challenge were found to exceed levels that could be accounted for by passive transudation of sICAM-1 from the circulation, the authors (35) suggested that the stimulatory action of IL-5 on sICAM release was attributed to a localized effect in the lungs. Our present extended observations provide evidence in support of the latter notion by demonstrating that exogenously administered IL-5 elicits a significant time-dependent increase in sICAM-1 release from cultured human ASM cells (Fig. 7). Thus when evaluated in light of our present results, it is conceivable that the reported IL-5-induced increase in the release of sICAM in asthmatic lungs may, at least in part, be related to a stimulatory effect of IL-5 on ASM cells. Clearly, to the extent that our present observations pertain to in vitro experimental conditions, the involvement of other cell types in the lung in contributing to IL-5-induced increases in sICAM-1 release in vivo remains to be identified.
A fundamental issue pertaining to the collection of findings in the
present study relates to the potential mechanism by which passive
sensitization of isolated ASM tissue and cultured ASM cells with human
atopic asthmatic serum induces ICAM-1/LFA-1 activation. Although this
mechanism remains to be elucidated, it is relevant to note that under
the same experimental conditions described herein, we previously
identified that the induced changes in ASM responsiveness obtained
after passive sensitization of rabbit ASM with atopic asthmatic serum
were initiated by the binding of IgE (present in the sensitizing serum)
to its low-affinity IgE receptor, FcRII (i.e., CD23), on the surface
of ASM cells (16). Furthermore, under these conditions, the changes in
ASM responsiveness in the atopic sensitized state were found to be mediated by the induced sequential autocrine release and action of IL-5
and IL-1
(19), an effect resulting in altered ASM responsiveness secondary to IL-1
-mediated upregulated expression and action of
Gi proteins, specifically G
i-2 and
G
i-3, which inhibit intracellular cAMP accumulation (17,
18). Given this earlier evidence, when examined in light of the present
observations, 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 CD23
activation in the ASM. Support for this speculated mechanism of action
is, in part, provided by the findings of recent studies demonstrating
that CD23 activation induces enhanced LFA-1-mediated adhesiveness in
CD4+ T cells and increased CAM expression (10) as well as a
Th2-type profile of cytokine release (11). The potential role and
mechanism of CD23-dependent activation of CAM expression and action in
airway smooth muscle remains to be systematically investigated.
In conclusion, the results of the present study provide new evidence demonstrating that pro-asthmatic-like changes in agonist constrictor and relaxant responsiveness in atopic asthmatic serum-sensitized ASM are attributed to ICAM-1/LFA-1 activation in the sensitized ASM itself. Moreover, the results demonstrate that ICAM-1/LFA-1 activation also elicits upregulated IL-5 protein release from sensitized ASM cells and that the latter phenomenon appears responsible for the associated increased expression and release of sICAM-1 by ASM cells in the atopic asthmatic sensitized state. Thus together with the conventional concept related to the interactive roles of specific CAMs and inflammatory cells in the pathobiology of asthma, the present findings identify a potentially important mechanism by which the resident ASM itself may, via autologous ICAM-1/LFA-1 activation, regulate its own state of altered responsiveness 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 |
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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.
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 22 November 1999; accepted in final form 13 January 2000.
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