Mechanism of cooperative effects of rhinovirus and atopic sensitization on airway responsiveness

Michael M. Grunstein, Hakon Hakonarson, Richard L. Hodinka, Neil Maskeri, Cecilia Kim, and Sing Chuang

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta . 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 Fcepsilon 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 Fcepsilon 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-1alpha , IL-1beta , IL-6, IL-8, IL-9, IL-11, tumor necrosis factor-alpha , 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-1beta by the RV-exposed ASM itself (14, 19). To the extent that these RV-mediated effects on IL-1beta 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, Fcepsilon 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 beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-4 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-1beta protein and expression of Fcepsilon RII mRNA as described in ELISA measurement of IL-1beta protein release and Determination of Fcepsilon RII mRNA expression in human ASM cells, respectively.

ELISA measurement of IL-1beta protein release. IL-1beta 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-1beta 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-1beta -specific secondary antibody is targeted first to an IL-1beta -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-1beta present in the culture medium.

Determination of Fcepsilon RII 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 Fcepsilon 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 Fcepsilon RII and RPL7 genes and included the following primer sets: 5'-CGTCTCTCAAGTTTCCAAG-3' (5' primer) and 5'-GCACTTCCGTTGGAATTTG-3' (3' primer) for Fcepsilon 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 Fcepsilon RII and RPL7 genes, respectively. The number of cycles was determined to be in the linear range of the PCR products. PCRs for the Fcepsilon 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 Fcepsilon RII and RPL7 DNA levels were assayed by Southern blot analysis with 32P-labeled probes prepared by pooling several RT-PCRs for the individual Fcepsilon 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 Fcepsilon RII protein expression in ASM tissue. Expression of cell surface Fcepsilon 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 Fcepsilon 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 Fcepsilon 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 Fcepsilon RII protein were quantitated by laser densitometry (Bio-Rad, Hercules, CA).

Reagents. The human ASM cells and SMBM were obtained from Clonetics. The Fcepsilon RII and RPL7 primers were obtained from Integrated DNA Technologies (Coralville, IA). The anti-ICAM-1, anti-CD11a, and anti-CD11b neutralizing antibodies; the IL-1beta ELISA kit; the mouse anti-human IL-1beta 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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ABSTRACT
INTRODUCTION
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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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Fig. 1.   Comparison of constrictor dose-response relationships to acetylcholine (ACh) in paired control serum-incubated, rhinovirus (RV)-inoculated, atopic asthmatic serum (AS)-sensitized, and AS-sensitized plus RV-inoculated (AS+RV) airway smooth muscle (ASM) tissue segments. Tmax, maximal isometric contractile force. Data are means ± SE from 8 paired experiments. Note that relative to control serum-incubated ASM, constrictor responses to ACh were significantly enhanced in the AS-sensitized, RV-inoculated, and AS+RV tissues.

In separate studies, during comparable levels of initial sustained ACh-induced contractions averaging ~50% of Tmax, cumulative dose-dependent ASM relaxation responses to the beta -adrenoceptor agonist isoproterenol were generated in the same tissues subjected to the above treatment conditions. As shown in Fig. 2, relative to control ASM, the Rmax responses to isoproterenol were significantly attenuated in the RV-exposed and in the atopic asthmatic serum-sensitized ASM segments (P < 0.01). Accordingly, relative to the Rmax value of 64.2 ± 3.7% obtained in the control ASM, the RV-exposed and atopic asthmatic serum-sensitized tissues yielded Rmax values averaging 51.2 ± 4.3 and 42.2 ± 5.0%, respectively, and the latter determinations further demonstrated that the attenuated relaxation responses to isoproterenol were significantly more pronounced in the atopic asthmatic serum-sensitized versus RV-exposed tissues (P < 0.05). The corresponding pD50 values for isoproterenol were also significantly reduced in the RV-exposed and the atopic asthmatic serum-sensitized ASM, averaging 6.11 ± 0.04 and 6.09 ± 0.05 -log M, respectively, relative to the control value of 6.35 ± 0.04 -log M (P < 0.05). Of note, in atopic asthmatic serum-sensitized tissues concomitantly exposed to RV (Fig. 2), the Rmax value was further attenuated at 33.1 ± 7.1%, representing a combined near-additive effect of the individual attenuated Rmax responses attributed to RV exposure and atopic asthmatic serum sensitization alone. Relative to the control value, the pD50 value obtained in the atopic asthmatic serum-sensitized plus RV-exposed ASM was reduced to 6.09 ± 0.07 -log M (P < 0.01), and the latter value was similar to those separately obtained in the selective RV-exposed and atopic asthmatic serum-sensitized tissue groups.


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Fig. 2.   Comparison of relaxation dose-response relationships to isoproterenol in paired control serum-incubated, RV-inoculated, AS-sensitized, and AS+RV ASM tissue segments. Data are means ± SE from 8 paired experiments. Note that relative to control serum-incubated ASM, relaxation responses to isoproterenol were significantly attenuated in the AS-sensitized and RV-inoculated tissues, and a near-additive attenuated effect on maximal relaxation was obtained in AS+RV ASM.

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 alpha -chain of the beta 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 beta 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 beta 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-1beta (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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Fig. 3.   Comparison of constrictor dose-response relationships to ACh in paired control serum-incubated and AS+RV ASM tissue segments in the absence and presence of anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody (MAb; A) and anti-CD11a MAb (B). Data are means ± SE from 6 paired experiments. Note that relative to control serum-incubated ASM, the heightened constrictor responses to ACh in AS+RV tissues were prevented by cotreatment of the tissue with either anti-ICAM-1 or anti-CD11a MAb.



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Fig. 4.   Comparison of relaxation dose-response relationships to isoproterenol in paired control serum-incubated and AS+RV ASM tissue segments in the absence and presence of anti-ICAM-1 MAb (A) and anti-CD11a MAb (B). Data are means ± SE from 6 paired experiments. Note that relative to control serum-incubated ASM, the attenuated relaxation responses to isoproterenol in AS+RV tissues were prevented by cotreatment of the tissues with either anti-ICAM-1 or anti-CD11a MAb.

Cooperative effects of RV and atopic sensitization on IL-1beta 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-1beta 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-1beta protein release from ASM. IL-1beta 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-1beta 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-1beta 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-1beta 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-1beta levels averaged 24.12 ± 5.27 and 16.88 ± 6.52 pg/g ASM, respectively, the augmented release of IL-1beta 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-1beta 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-1beta 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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Fig. 5.   Comparison of interleukin (IL)-1beta protein elaboration into the culture medium of rabbit ASM tissues (A) and human ASM cells (B) after 24 h of incubation with control serum and atopic asthmatic serum in both the absence and presence of RV. Note that relative to control serum-incubated ASM tissues and cells, release of IL-1beta protein was significantly increased in the AS-sensitized and RV-inoculated ASM preparations, and this effect was potentiated by combined AS sensitization and RV inoculation of the tissue and cell preparations. Moreover, enhanced release of IL-1beta was prevented by cotreatment of AS+RV ASM tissues and cells with anti-CD11b MAb. Data are means ± SE from 5 paired experiments with ASM tissues and 6 paired experiments with cultured human ASM cells.

Modulatory effects of RV on Fcepsilon RII 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, Fcepsilon 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 Fcepsilon RII. Analysis of Fcepsilon 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 Fcepsilon 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 Fcepsilon 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 Fcepsilon RII and corresponding RPL7 signals, the intensity of the Fcepsilon RII mRNA signal was progressively enhanced at 3, 6, and 24 h after exposure of the cells to RV.


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Fig. 6.   Comparison of Fcepsilon RII mRNA expression in cultured human ASM cells after 0-, 3-, 6-, and 24-h exposures to vehicle alone (control) and to RV16. Constitutive expression of ribosomal protein (RP) L7 mRNA was used to control for gel loading. The blots were probed with human-specific Fcepsilon RII and RPL7 32P-labeled cDNA probes (see METHODS). Note that relative to the faint and unaltered Fcepsilon RII mRNA signals detected in control cells, mRNA expression of Fcepsilon RII was progressively enhanced at the indicated times after exposure of cells to RV. The intensities of the mRNA signal for the constitutively expressed RPL7 gene were essentially unaltered under both treatment conditions.

In separate studies, we further examined whether cell surface protein expression of Fcepsilon RII in rabbit ASM tissue was also modulated in the presence of RV. Western immunoblot analysis of Fcepsilon RII protein expression was conducted in membrane homogenates of ASM tissues exposed for 24 h to either control or atopic asthmatic serum in both the absence and presence of RV. As depicted by a representative experiment in Fig. 7, in the control serum-incubated state, Fcepsilon RII expression was distinctly increased in the RV-inoculated ASM as quantified by laser densitometry at 8.1-fold above the expression level detected in the corresponding noninoculated ASM. In further comparison with the noninoculated control ASM, Fcepsilon RII expression was also significantly increased by 4.9-fold in the noninoculated atopic asthmatic serum-sensitized ASM. Finally, in atopic asthmatic serum-sensitized tissues exposed to RV, the level of Fcepsilon RII expression was most markedly enhanced and amounted to >20- and 10.2-fold above the levels detected in the control noninoculated and RV-inoculated ASM tissues, respectively.


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Fig. 7.   Representative Western blots depicting Fcepsilon RII protein expression in membrane homogenates isolated from rabbit ASM tissue samples incubated for 24 h in control (C) and AS serum in the absence and presence of RV inoculation. Note that relative to the corresponding control serum-incubated tissues, ASM incubated in AS serum exhibited significantly increased Fcepsilon RII expression in both the noninoculated and RV-inoculated states. No. on right, molecular mass.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta in the RV-exposed ASM itself (14), and both the induction of IL-1beta 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-1beta 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-1beta release; 2) the latter phenomena are associated with RV-induced upregulation of the expression of the low-affinity receptor for IgE, Fcepsilon 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 beta 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 beta 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-1beta 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-1beta in the ASM itself. Under these circumstances, the action of IL-1beta was found to be largely mediated by its induced enhanced expression and activation of Gi protein (specifically, Galpha i-2 and Galpha 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-1beta 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-1beta 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 Fcepsilon RII 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 Fcepsilon RII activation in the sensitized ASM. Support for the latter speculated mechanism of action is, in part, provided by recent reports demonstrating that Fcepsilon 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 Fcepsilon RII mRNA (Fig. 6) and Fcepsilon 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 Fcepsilon RII expression in ASM is upregulated in the atopic sensitized state and that this effect is attributed to activation of the endogenously expressed Fcepsilon RII receptor itself by the elevated IgE present in the atopic sensitizing serum. Given these earlier observations, together with the finding that activation of Fcepsilon 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 Fcepsilon 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 Fcepsilon RII protein expression beyond that elicited by exposure of the tissue to the atopic sensitizing serum alone (Fig. 7). Insofar as Fcepsilon 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-1beta 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 Fcepsilon 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.


    ACKNOWLEDGEMENTS

We thank J. S. Grunstein for expert technical assistance and M. Brown for typing the manuscript.


    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.


    REFERENCES
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RESULTS
DISCUSSION
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