Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1beta

Hakon Hakonarson1, Carrie Carter1, Neil Maskeri1, Richard Hodinka2, and Michael M. Grunstein1

Divisions of 1 Pulmonary Medicine and 2 Immunologic and Infectious Diseases, Joseph Stokes, Jr. Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An important interplay exists between specific viral respiratory pathogens, most commonly rhinovirus (RV), and altered airway responsiveness in the development and exacerbations of asthma. Given that RV infection reportedly induces the release of various cytokines in different cell types and that the reported effects of RV on airway smooth muscle (ASM) responsiveness are highly comparable to those obtained in ASM exposed to the proinflammatory cytokine interleukin (IL)-1beta , this study examined whether RV (serotype 16)-mediated pertubations in ASM responsiveness are mechanistically coupled to altered induced expression and action of IL-1beta in RV-exposed isolated rabbit and human ASM tissue and cultured cells. Relative to control tissues, ASM inoculated with RV exhibited significantly increased maximal isometric contractility to ACh (P < 0.01) and attenuated relaxation to isoproterenol (P < 0.005). In extended studies, we found that 1) the RV-induced changes in ASM responsiveness were ablated by pretreating the tissues with the IL-1 recombinant human receptor antagonist; 2) in contrast to their respective controls, RV-inoculated ASM tissue and cultured cells exhibited progressively induced expression of IL-1beta mRNA and elaboration of IL-1beta protein at 6 and 24 h after viral exposure; and 3) the latter effect of RV was inhibited in the presence of a monoclonal antibody to intercellular adhesion molecule-1, the endogenous receptor for most RV. Collectively, these observations provide new evidence demonstrating that "pro-asthmatic-like" pertubations in agonist responsiveness elicited in RV-exposed ASM are largely attributed to the induced autologous expression and autocrine action of IL-1beta in the virus-infected ASM.

cholinergic contractility; beta -adrenoceptor relaxation; viral respiratory infection; cytokine signaling; airway reactivity; asthma


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS CLINICALLY WELL established that rhinovirus (RV) infection is the most common trigger of respiratory exacerbations in individuals with asthma (2, 9, 10, 21, 29). Moreover, a transient airway hyperreactivity is also reported to occur in nonasthmatic subjects after respiratory infections with RV (1, 9). Although the basic mechanism(s) underlying RV-induced asthma exacerbation or transient airway hyperreactivity remains to be established, there is ample evidence that infection of various cell types with RV (e.g., respiratory epithelium, fibroblasts, monocytes, and macrophages) induces the expression and release of different cytokines, including interleukin (IL)-1beta , IL-6, IL-8, IL-9, IL-11, and tumor necrosis factor (TNF)-alpha (19, 30, 32, 37). These findings suggest that specific cytokines may play an important role in the pathogenesis of symptomatic RV infections. In support of this concept, we recently demonstrated that exposure of airway smooth muscle (ASM) tissue to specific proinflammatory cytokines, most notably IL-1beta , produces changes in the tissue's responsiveness that closely resemble the characteristic "proasthmatic" ASM phenotype, including heightened ASM constrictor responsiveness and attenuated ASM relaxation responsiveness to specific receptor/G protein-coupled agonists (16). Moreover, comparable changes in responsiveness were observed in ASM tissues passively sensitized with human atopic asthmatic serum, wherein the latter changes were attributed to the autologously induced upregulated expression, extracellular release, and autocrine action of IL-1beta by the ASM itself (17, 18). In light of this evidence, the present study examined whether infection of ASM with RV elicits changes in constrictor and relaxant responsiveness that are mechanistically coupled to the induced expression and autocrine action of IL-1beta . The results provide new evidence that 1) RV exposure of isolated ASM produces heightened contractility to ACh and attenuated relaxation to beta -adrenergic receptor stimulation with isoproterenol; 2) these effects are largely attributed to RV-induced autologous expression of IL-1beta and its resultant autocrine action in the RV-infected ASM; and 3) the autocrine release and effect of IL-1beta in the RV-exposed state is triggered by the binding of RV to its endogenous receptor, intercellular adhesion molecule (ICAM)-1.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals. Adult New Zealand White rabbits were used in this study, which was approved by the Biosafety and Animal Research Committee of the Joseph Stokes Research Institute at Children's Hospital of Philadelphia. The animals had no signs of respiratory disease for several weeks before the study.

Culture of respiratory viruses. Virus stock solutions were generated by infecting monolayer cultures of sensitive cell systems with freshly isolated respiratory viruses from the Clinical Virology Laboratory at the Children's Hospital of Philadelphia, including human RV serotype 16 (RV16) and adenovirus. Human embryonic lung fibroblasts (MRC-5) were used to culture the RV, and human carcinoma cells from lung (A549) were used to culture adenovirus. The cultures were grown in modified MEM supplemented with Earle's balanced salt solution, 1% L-glutamine, 7.5% FBS, HEPES buffer, and antimicrobial agents (5 µg/ml gentamicin, 10 µg/ml vancomycin, and 10 µg/ml amphotericin B). When infection was notably advanced, as evidenced by obvious cytopathic effects (CPE), cell supernatants were harvested and frozen in aliquots (~2-4 × 106 virus particles/aliquot) at -70°C, as previously described (14), for use in the experiments described below.

Detection of respiratory viruses in ASM cells. Rabbit ASM cells cultured in our laboratory (see below) were propagated in 16 × 125-mm sterile plastic tissue culture dishes using the above complete medium. Confluent monolayers of ASM cells were then separately inoculated with stock solutions (~1 × 106 virus particles/T25 flask) of the above viruses as previously described in the laboratory. Detection of infection by adenovirus was confirmed by immunofluorescence staining of acetone-fixed infected cells using mouse monoclonal antibodies directed against adenovirus and a goat anti-mouse FITC conjugate. Due to the lack of commercially available antibodies to RV serotypes, detection of infection of the ASM cells with RV was confirmed by RT-PCR and Southern blotting. The PCRs were performed using cDNA reverse transcribed from total RNA isolated from ASM cells that were inoculated for 7 days with RV, using RV-specific primer pairs (i.e., 5' primer 5'-GCACTTCTGTTTCCCC-3' and 3' primer 5'-CGGACACCCAAAGTAG-3') and a cDNA probe prepared by pooling, purifying, and sequencing the above PCR reactions.

Viral inoculation of ASM tissue. Our method for preparing rabbit ASM tissue has been previously described (17). Briefly, after anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg), the animals were killed with an intravenous overdose of pentobarbital sodium (130 mg/kg). The tracheae were removed, scraped of loose connective tissue and epithelium, and then divided into 6- to 8-mm-ring segments. In comparable studies, human bronchial smooth muscle tissue was also obtained from patients undergoing lung resection for peripheral lung carcinoma and having no evidence of obstructive lung disease as assessed by routine pulmonary function testing preoperatively. After lung resection, the specimens were macroscopically examined and, from tumor-free sites, bronchi (3-5 mm ID) from the 3rd to 5th generations were dissected free from the surrounding parenchyma and sectioned into ring segments. Each alternate adjacent human or rabbit ASM ring segment was then incubated for 90 min at the optimal replication temperature for each virus in DMEM in the absence and presence of maximum effective concentrations of either RV or adenovirus (~1 × 106 viral particles/ml). The tissues were then further incubated for 24 h at room temperature. The viral inoculation experiments were conducted in the absence and presence of 1 h of pretreatment with the recombinant human IL-1 receptor antagonist (IL-1ra; 140 ng/ml). The tissues were aerated with a supplemental O2 mixture (95% O2-5% CO2) throughout the incubation period, and, thereafter, the tissue responsiveness to specific ASM constrictor and relaxant agonists was compared as described below. In extended experiments, IL-1beta mRNA and protein expression were examined in the human ASM tissue samples that were inoculated with RV16 or vehicle alone in the absence and presence of anti-ICAM-1 monoclonal antibody (MAb; 4 µg/ml) as described below.

Viral inoculation of cultured ASM cells. Rabbit ASM cells cultured in our laboratory have been previously characterized in detail with respect to their distinguishing morphological, histological, and immunological features (23). The cell isolation and subcultivation procedures were previously described (23). Briefly, ASM cells were isolated from epithelium-denuded trachealis muscle from adult New Zealand White rabbits. After digestion in Ham's F-12 containing 30 µg/ml protease, 55 µg/ml type IV collagenase, and 100 µg/ml trypsin inhibitor, the dissociated cells were centrifuged and resuspended in Ham's F-12 containing 10% FBS and 100 µg/ml gentamicin sulfate. The cells were then inoculated in 100-mm tissue culture dishes, and, after 4 wk, the cells had sufficiently proliferated to permit routine subcultivations. At weekly intervals, the subcultivated cells were suspended and then inoculated at a density of 1 × 104 cells/cm2 in 75-cm2 tissue culture flasks containing Ham's F-12 with 10% FBS and were incubated at 37°C in a humidified atmosphere of 5% CO2-95% air. When the cells were >90% confluent, the original culture medium was replaced with Ham's F-12 for 24 h. The medium was then aspirated, and the cells were inoculated, in separate experiments, with maximum effective concentrations of either RV or adenovirus (~1 × 106 virus particles/T25 flask) in 0.5 ml of culture medium for 60 min at the optimal replication temperature for each virus in the absence and presence of anti-ICAM-1 MAb (4 µg/ml). The cells were then washed one time with fresh medium and incubated at 37°C for various time points. The cells were then prepared for detection of IL-1beta mRNA and protein expression as described below.

In parallel experiments, human bronchial smooth muscle cells (Clonetics, San Diego, CA) derived from two male donors without lung disease, aged 16 and 21 yr, were grown in smooth muscle basal medium (SMBM) supplemented with 5% FBS, 5 ng/ml insulin, 10 ng/ml epidermal growth factor, 2 ng/ml fibroblast growth factor, 50 ng/ml gentamicin, and 50 ng/ml amphotericin B. The standard experimental protocol involved growing the cells to confluence in the above medium and then starving the cells in unsupplemented SMBM for 24 h, at which time the cells were treated with either RV or adenovirus in the absence and presence of anti-ICAM-1 MAb. The cellular RNA was harvested at the various time points and used for detection of IL-1beta mRNA, and the cell medium was salvaged for determination of IL-1beta protein release as described below.

Pharmacodynamic measurements of ASM responsiveness. After incubation of the tissue preparations, each rabbit airway segment was suspended longitudinally between stainless steel triangular supports in siliconized Harvard 20-ml organ baths. The lower support was secured to the base of the organ bath, and the upper support was attached via a gold chain to a Grass FT.03C force transducer from which isometric tension was continuously displayed on a multichannel recorder. Care was taken to place the membranous portion of the trachea between the supports to maximize the recorded tension generated by the contracting trachealis muscle. The tissues were bathed in modified Krebs-Ringer solution containing (in mM) 125 NaCl, 14 NaHCO3, 4 KCl, 2.25 CaCl2 · H2O, 1.46 MgSO4 · H2O, 1.2 NaH2PO4 · H2O, and 11 glucose. The baths were aerated with 5% CO2 in O2; a pH of 7.35-7.40 was maintained, and the organ bath temperature was held at 37°C. Passive resting tension of each tracheal smooth muscle (TSM) segment was set at 2.0 g after the tissue had been passively stretched to a tension of 8 g to optimize the resting length of each segment as previously described in our laboratory (14). The tissues were allowed to equilibrate in the organ baths for 45 min, at which time each tissue was primed with a 1-min exposure to 10-4 M ACh. Cholinergic contractility was subsequently assessed in the TSM segments by cumulative administration of ACh in final bath concentrations ranging from 10-10 to 10-3 M. Thereafter, in separate studies, relaxation dose-response curves to isoproterenol (10-10 to 10-4 M) were conducted in tissues half-maximally contracted with ACh. The relaxant responses to isoproterenol were analyzed in terms of percent maximal relaxation (Rmax) from the initial level of active cholinergic contraction, and sensitivity to the relaxing agent was determined as the negative logarithm of the dose of isoproterenol producing 50% of Rmax (pD50; i.e., geometric mean ED50 value).

Determination of IL-1beta expression in ASM cells by RT-PCR and Southern blot analysis. Total RNA was isolated from rabbit and human ASM cells in the absence and presence of exposure of the cells to RV16 for varying durations using the modified acid guanidinium thiocyanate phenol-chloroform extraction method to include proteinase K (in 0.5% SDS) digestion of protein in the initial RNA pellet (15). The concentration of each RNA sample was then determined spectrophotometrically. This procedure consistently yielded 15-25 µg of intact RNA for each T75 flask of ASM cells. To analyze the mRNA expression of IL-1beta , we used an RT-PCR protocol and IL-1beta primer pairs based on the published sequences of the rabbit (3) and human (6) IL-1beta genes and included the following primer sets: 1) rabbit-specific 5'-GCACCTCTCAGACAGAGTAC-3' and 5'-GTGGTTGCTGATAGAAGCTG-3' and 2) human-specific 5'-AGATGAAG TGCTCCTTCCAG-3' and 5'-CAACACGCAGGACAGGTACAG-3'. To control for the transcription levels of the samples, we used 1) the rabbit-specific alpha -actin primers 5'-CGACATCAAGGAGAAGCTG-3' and 5'-CTAGAAGCATTTGCGGTGC-3' and 2) the human-specific ribosomal protein L7 (RPL7) primers 5'-AAGAGGCTCTCATTTTCCTGGCTG-3' and 5'-TCCGTTCCTCCCCATAATGTTCC-3', based on the published sequences of the rabbit alpha -actin (25) and human RPL7 (28) genes. cDNA was synthesized using 2.5 µg of total RNA isolated from cells after 0, 6, and 24 h exposure to RV or medium alone. The cDNA was primed with oligo(dT)12-18, and 2 µl of cDNA were used for each PCR. The cycling profile used was as follows: denaturation, 95°C for 1 min; annealing, 52°C for 1.0 min, and extension, 72°C for 1 min with 35 cycles for the human and rabbit IL-1beta gene and 24 cycles for the alpha -actin and RPL7 genes. The number of cycles was determined to be within the linear range of the PCR products. Equal aliquots of each PCR reaction were then run on a 1.2% agarose gel and subsequently transferred to a zeta probe membrane overnight in 0.4 N NaOH. After capillary transfer, the DNA was immobilized by ultraviolet (UV) cross-linking using a Stratalinker UV cross-linker 2400 at 120,000 µJ/cm2 (Stratagene). Prehybridization in a Techne hybridization oven was conducted for 2-3 h at 42°C in 50% formaldehyde, 7% (wt/vol) SDS, 0.25 M NaCl, 0.12 M Na2HPO4 (pH 7.2), and 1 mM EDTA. Hybridization was for 20 h at 42°C in the same solution. The IL-1beta , alpha -actin, and RPL7 DNA levels were assayed by Southern blot analysis using 32P-labeled probes, including the rabbit-specific 358-bp IL-1beta and 415-bp alpha -actin and human-specific 471-bp IL-1beta and 157-bp RPL7 probes, 32P-labeled in random primer reactions. The probes were prepared by pooling several RT-PCRs for the PCR fragments, purifying them from a 1.2% agarose gel using a Qiaex II agarose gel extraction kit, and then sequencing. Washes were as follows: 1 × 15 min in 2× saline-sodium citrate (SSC), 0.1% SDS; 1 × 15 min in 0.1× SSC-0.1% SDS both at room temperature; and 2 × 15 min at 50°C in 0.1× SSC-0.1% SDS. Southern blots were quantified by direct measurements of radioactivity in each band using a phosphorimager (Molecular Dynamics).

Determination of IL-1beta expression in human ASM cells by Northern blot analysis. Total RNA isolated from the cultured human ASM cells maintained in SMBM in the absence and presence of 0, 6, and 24 h exposure to RV was also fractionated (15 µg/lane) in 1% agarose and 2.2 M formaldehyde denaturing gels. After capillary transfer to zeta probe membranes (Bio-Rad) in 10× SSC (1× SSC = 0.015 M sodium citrate), RNA was immobilized by UV cross-linking using a Stratalinker UV cross-linker 2400 at 120,000 µJ/cm2. After prehybridization and hybridization, the IL-1beta mRNA levels were examined by Northern blot analysis using the human-specific IL-1beta probe as described above. A human-specific probe for constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control for RNA loading.

ELISA measurements of IL-1beta protein. IL-1beta protein levels were measured in the culture medium from human ASM tissues and ASM cultured cells after 0, 6, and 24 h exposure to RV16 by means of a commercially available enzyme-specific immunoassay. As per the manufacturer's protocol, the assay was performed using a double-antibody sandwich strategy in which an ACh esterase (AChE), Fab-conjugated IL-1beta -specific secondary antibody, is targeted to a first cytokine-captured antibody. The enzymatic activity of the AChE was measured spectrophotometrically, and, relative to a linear standard curve (range: 0-250 pg/ml), the results were used to quantify the amount of the targeted IL-1beta present in the cell and tissue culture media.

Statistical analysis. Unless otherwise indicated, results are expressed as mean values ± SE. Statistical analysis was performed by means of the two-tailed paired Student's t-test. P values <0.05 were considered significant.

Reagents. The human IL-1beta , rabbit alpha -actin, and RV-specific primers were obtained from Integrated DNA Technologies (Coralville, IA). The IL-1beta ELISA kit and IL-1ra were obtained from R&D Systems (Minneapolis, MN). The ICAM-1 MAb, ACh, and isoproterenol hydrochloride were obtained from Sigma Chemical (St. Louis, MO). All drug concentrations are expressed as final bath concentrations. Isoproterenol and ACh were made fresh for each experiment, dissolved in normal saline to prepare 10-4 and 10-3 M solutions, respectively. The human tissue was provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RV-induced changes in ASM responsiveness. Detection of infection of ASM cells by adenovirus was confirmed by evidence of CPE on days 3, 7, and 14 postinoculation. CPE were detected using immunofluorescence staining of acetone-fixed infected cells with a mouse MAb directed against adenovirus and a goat anti-mouse FITC conjugate (Fig. 1A). Due to the lack of commercially available antibodies to RV serotypes, as depicted in Fig. 1B, infection of ASM cells with RV16 was confirmed by RT-PCR and Southern blotting using RV-specific primer pairs (see MATERIALS AND METHODS) and a cDNA probe prepared by purifying and sequencing the above PCRs.


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Fig. 1.   Detection of rhinovirus (RV) and adenovirus infection in cultured airway smooth muscle (ASM) cells. A: demonstration of viral antigen to adenovirus in rabbit ASM cells cultured in supplemented MEM. Experimental cells ( passages 6-8) were stained with virus-specific antibodies after 72 h of infection (see MATERIALS AND METHODS). Viral antigen to adenovirus was readily detectable in infected cells. Note: adenovirus produced classical cytopathic effects (CPE) as demonstrated by the various sizes of rounded refractile cells with characteristic lytic lesions. B: Southern blot of RV mRNA expression in rabbit ASM cells. Total RNA was isolated from cells inoculated for 0, 48, and 72 h in the absence (control) and presence (RV treated) of RV exposure. RNA isolated from RV stock solution was used as a positive control (RV cDNA). Blot was probed with a sequenced [32P]cDNA probe prepared from pooled purified RT-PCR reactions for the RV gene. Note: in contrast to lack of expression in control cells, RV mRNA expression was induced in RV-infected ASM cells at 48 and 72 h.

To determine the role of IL-1beta in mediating any induced changes in ASM responsiveness in the RV-infected state, airway constrictor and relaxation responses were separately examined in isolated TSM segments that were exposed to either RV16 or vehicle alone in the absence and presence of the endogenous IL-1ra. In contrast to adenovirus inoculation, which had no effect relative to control tissues incubated with vehicle alone, the maximal constrictor (Tmax) responses to ACh were significantly enhanced in TSM that were exposed to RV16 (Fig. 2). Accordingly, the mean ± SE Tmax values amounted to 99.08 ± 7.4 and 124.69 ± 21.00 g/g TSM wt in the control and RV-exposed tissues, respectively (P < 0.01); and the corresponding pD50 (i.e., -log ED50) values averaged 4.70 ± 0.08 and 5.13 ± 0.15 -log M, respectively (P < 0.05). This induced augmented constrictor responsiveness to ACh, however, was largely prevented in RV-exposed tissues that were pretreated with IL-1ra (Fig. 2), wherein the mean ± SE Tmax and pD50 values amounted to 108.58 ± 15.39 g/g TSM wt and 4.89 ± 0.12 -log M, respectively. There were no significant differences between the latter Tmax and pD50 values obtained in the IL-1ra-pretreated ASM and the corresponding determinations obtained in control tissues, although the values were significantly different (P < 0.05) from those obtained in the presence of RV alone. Moreover, in contrast to RV-exposed ASM, pretreatment with IL-1ra had no effect in adenovirus-exposed or control (vehicle-treated) ASM tissues (data not shown).


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Fig. 2.   Constrictor dose-response relationships to ACh. Comparison of constrictor dose-response relationships to ACh in control (open circle ) and RV-exposed ASM in the absence () and presence () of interleukin (IL)-1 receptor antagonist (ra). Data are mean values ± SE from 6 separate experiments. Note: both the maximal constrictor and pD50 values to ACh are significantly enhanced (P < 0.01) in tissues exposed to RV, and these effects of RV are largely inhibited in the presence of IL-1ra.

In separate studies, during comparable levels of initial sustained ACh-induced contractions in RV-exposed and control airway segments, averaging ~45% of Tmax, administration of the beta -adrenergic receptor agonist isoproterenol elicited cumulative dose-dependent relaxation of the precontracted TSM segments. Relative to control TSM, however, Rmax and pD50 values to isoproterenol were significantly attenuated in the RV-exposed TSM (Fig. 3). Accordingly, the mean Rmax values for isoproterenol amounted to 56.19 ± 7.50% in the RV-exposed tissues compared with 73.03 ± 4.10% in the control TSM (P < 0.05); and the corresponding pD50 values averaged 6.28 ± 0.08 and 6.59 ± 0.16 -log M, respectively (P < 0.05). Furthermore, as also depicted in Fig. 3, the attenuated isoproterenol-induced relaxation responses were largely ablated in RV-exposed TSM that were pretreated with IL-1ra, wherein the mean Rmax value for isoproterenol amounted to 73.64 ± 5.68% and the corresponding pD50 value averaged 6.51 ± 0.13 -log M. There were no significant differences between the latter Rmax and pD50 values and those obtained in control TSM, although the values were significantly different (P < 0.05) from those obtained in the presence of RV alone. Finally, in contrast to RV, exposure of ASM to adenovirus had no effect on the tissue's relaxant responsiveness to isoproterenol (data not shown).


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Fig. 3.   Relaxation response curves to isoproterenol. Comparison of relaxation response curves to isoproterenol in control (open circle ) and RV-exposed ASM in the absence () and presence () of IL-1ra. Data are mean values ± SE from 6 separate experiments. Note: both the maximal relaxation and pD50 values to isoproterenol are significantly attenuated in RV-exposed ASM, and these effects of RV are largely inhibited in the presence of IL-1ra.

IL-1beta mRNA expression in RV-exposed ASM. Respiratory infections with RV have been associated with the induced expression and elaboration of specific cytokines in both natural and experimental human and animal studies involving RV infection (19, 30, 32, 37). Given our above findings, and to the extent that we recently identified a critical role for IL-1beta in mediating changes in agonist responsiveness in atopic asthmatic serum-sensitized ASM that are similar to the above effects obtained after RV inoculation, we next examined whether exposure of ASM cells to RV16 is associated with an induced altered expression and action of IL-1beta . Accordingly, in initial studies, using RT-PCR and rabbit-specific IL-1beta primers, cDNA was reverse transcribed from total RNA isolated from cultured rabbit or human ASM cells and primed with oligo(dT), and Southern blots were probed with cDNA probes specific for rabbit and human IL-1beta (see MATERIALS AND METHODS). A 415-bp alpha -actin or 157-bp RPL7 probe was also used to control for gel loading, and the signals for the IL-1beta , alpha -actin, or RPL7 PCR products were quantified using a phosphorimager. As demonstrated in Fig. 4, in contrast to the unaltered constitutive expression of alpha -actin or RPL7, the IL-1beta signal was significantly enhanced in both the rabbit (Fig. 4A) and human (Fig. 4B) ASM cells that were exposed to RV at 6 and 24 h, whereas the corresponding expression of IL-1beta mRNA was essentially unaltered in control cells that were treated with vehicle alone. Qualitatively similar results were also obtained by Northern blot analysis of IL-1beta mRNA expression in cultured human ASM cells that were exposed to RV compared with vehicle alone (data not shown). Finally, unlike IL-1beta mRNA, we found no evidence of either constitutive or induced expression of TNF-alpha in either rabbit or human ASM cells under control or RV-exposed conditions (data not shown).


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Fig. 4.   Regulation of IL-1beta mRNA expression by RV. Representative Southern blots of IL-1beta mRNA expression in rabbit (A) and human (B) ASM cells after 0, 6, and 24 h inoculation in cell culture medium in the absence (control) and presence (RV exposed) of RV serotype 16. Note: in contrast to unaltered alpha -actin (A) and ribosomal protein L7 (RPL7; B) expression, expression of IL-1beta was significantly induced at 6 and 24 h in cells that were exposed to RV. Blots were probed with sequenced [32P]cDNA probes prepared from pooled purified RT-PCR reactions for the rabbit IL-1beta and alpha -actin and the human IL-1beta and RPL7 genes, respectively.

IL-1beta protein release by RV-exposed ASM. In light of the above observations, additional experiments were conducted to evaluate the effects of RV exposure of human ASM cells and ASM tissue on the release of IL-1beta protein in the cell/tissue culture medium, examined using an IL-1beta -specific immunoassay. There were virtually undetectable levels of IL-1beta in the culture medium of either the ASM tissue or ASM cultured cell preparations exposed to vehicle alone. In contrast to control conditions, as exemplified in Fig. 5, ASM cells exposed to RV depicted progressively enhanced elaboration of IL-1beta protein in the culture medium for up to 24 h. A qualitatively similar effect of RV was obtained in the ASM tissue preparations. Moreover, as depicted in Fig. 6, the stimulated release of IL-1beta protein obtained at 24 h in the RV-exposed ASM tissue (Fig. 6A) and cultured ASM cells (Fig. 6B) was markedly inhibited by pretreating the tissue or cell preparations with a monoclonal blocking antibody to ICAM-1, the endogenous receptor for the vast majority of RV (13, 35). Thus, taken together, the above results demonstrate that exposure of ASM tissue or cultured ASM cells to RV induces the mRNA expression and elaboration of IL-1beta protein in the RV-infected state, a phenomenon attributed to binding of RV to its ICAM-1 receptor.


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Fig. 5.   RV-induced release of IL-1beta in cultured human ASM cells. Comparison of levels of IL-1beta protein released in the culture medium of cultured human ASM cells after 0, 6, and 24 h exposure to vehicle alone (open circle ) or to RV serotype 16 (). Note: in contrast to exposure to vehicle alone, which had no effect, cells exposed to RV depicted progressively enhanced elaboration of IL-1beta in the culture medium for up to 24 h. Data are mean values from 2 separate experiments.



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Fig. 6.   Inhibitory effects of anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody (MAb) on RV-induced release of IL-1beta . Comparison of levels of IL-1beta protein released in culture medium of human ASM tissues (A) and cultured human ASM cells (B) after 24-h exposure to vehicle (i.e., controls) alone (open bars) or RV serotype 16 in the absence (filled bars) and presence (hatched bars) of anti-ICAM-1 MAb. Note: in contrast to exposure to vehicle alone, which had no effect, induced elaboration of IL-1beta protein measured in the ASM tissue (A) and cell culture (B) media at 24 h was markedly attenuated by pretreatment with anti-ICAM-1 MAb. Data are mean values from 2 separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Viral respiratory infections are often associated with acute episodes of wheezing and asthma exacerbations throughout childhood. Beyond infancy, RV represents the predominant common cold virus that has been implicated in triggering acute wheezing in asthmatic children and adults. In this connection, RV infection has been associated with increases in constrictor agonist-mediated airway reactivity (1, 2, 10, 21), increased influx of eosinophil and other inflammatory cells in the respiratory tract (9), and the synthesis and elaboration of a host of cytokines in both natural and experimental human and animal studies involving RV infection (7, 19, 30, 33). A priori, when taken together, these effects of RV infection closely parallel the known proinflammatory changes in airway function observed in atopic asthma and, hence, suggest that a potentially important common mechanism underlies the perturbed airway function seen both in asthma and after RV infection. In this regard, it is relevant to note that we recently identified that the perturbations in ASM constrictor and relaxant responsiveness experimentally induced in naive ASM after its passive sensitization with human atopic asthmatic serum were attributed to the autocrine release and action of IL-1beta by the sensitized ASM itself (16). Given the latter, the present study examined the potential role of IL-1beta in also mediating RV-induced perturbations in ASM responsiveness. Our results demonstrated that 1) RV-exposed ASM displays heightened ASM contractility to ACh and impaired relaxation responsiveness to beta -adrenoceptor stimulation; 2) these changes in ASM responsiveness obtained in RV-exposed ASM are prevented by blockade of IL-1beta action; 3) exposure of ASM tissue or cultured cells to RV induces the autologous mRNA expression and elaboration of IL-1beta protein by the ASM; and 4) the latter is attributed to the binding of RV to its endogenous receptor, ICAM-1. Collectively, these observations provide new evidence that the altered responsiveness of RV-exposed ASM is largely due to the induced expression of IL-1beta by the virally infected ASM and that the latter cytokine acts in an autocrine fashion to elicit enhanced ASM constrictor responsiveness and impaired ASM relaxation.

Our above present findings closely resemble those recently reported in isolated rabbit ASM passively sensitized with human atopic asthmatic serum, wherein we found that comparable changes in ASM responsiveness (i.e., increased contractility and attenuated relaxation) were mechanistically coupled to the induced expression and autocrine action of IL-1beta protein in the atopic sensitized ASM (18). Moreover, in this regard, the latter IL-1beta -mediated changes in ASM responsiveness obtained in atopic asthmatic serum-sensitized tissues (18), as well as in naive ASM treated with exogenously administered IL-1beta (16), were found to be due to IL-1beta -induced enhanced expression and function of Gi protein (16, 17). The latter is associated with inhibition of adenylate cyclase activity and, to a lesser extent, with stimulation of phospholipase C (8, 27). Accordingly, these Gi protein-mediated effects result in both attenuated beta -adrenoceptor-coupled ASM relaxation, secondary to reduced accumulation of cAMP, and increased constrictor agonist-coupled ASM contractility, secondary to enhanced accumulation of the Ca2+-mobilizing second messenger inositol 1,4,5-trisphosphate (26). In light of this evidence, it is reasonable to assume that our observed IL-1beta -mediated changes in ASM responsiveness after RV exposure were likely related to IL-1beta -induced upregulated Gi protein effects on receptor-coupled transmembrane signaling regulating ASM contraction and relaxation.

ICAM-1 is known to be the principal receptor for the vast majority (i.e., >90%) of RV subtypes (13, 35). Apart from its latter function, ICAM-1 activation has also been proposed in mediating the airway eosinophilia and hyperresponsiveness obtained in a primate model after antigen challenge because both the eosinophil influx and airway hyperresponsiveness were attenuated by the administration of an anti-ICAM-1 antibody (4, 15, 36). Whereas the latter study implicated a role for ICAM-1 activation that is coupled to eosinophil influx in mediating the altered airway responsiveness, a more recent study conducted in antigen-sensitized Brown Norway rats showed that administration of an anti-ICAM-1 antibody was found to attenuate the animal's airway constrictor hyperresponsiveness without producing a concomitant reduction in the degree of airway inflammation (31). Thus, although ICAM-1 activation may contribute to the development of airway constrictor hyperresponsiveness, the mechanism underlying this phenomenon may not be entirely dependent on inflammatory cell influx.

In extending the above concept, it is relevant to note that expression of ICAM-1 has been demonstrated in lung stromal cells, including endothelial cells, epithelial cells, and ASM cells, and that ICAM-1 expression in these cell types is upregulated after exposure of asthmatic subjects to allergen (20, 22, 24, 33, 34). Moreover, recent studies have demonstrated that different viral respiratory pathogens, including RV, can directly infect lung stromal cells (e.g., fibroblasts) and thereby induce the elaboration of specific cytokines (7). Collectively, this evidence, when considered in light of our present finding that the administration of anti-ICAM-1 MAb to ASM tissue and cultured cells largely inhibited RV-induced IL-1beta release (Fig. 6), suggests a critical role for RV/ICAM-1-coupled activation of ASM in autologously mediating the observed changes in ASM responsiveness, secondary to the enhanced release and autocrine action of IL-1beta . Thus the findings of the present study, together with the above recent evidence, support the emerging general concept that, notwithstanding the contemporary view that the effects of RV on airway responsiveness likely occur secondary to infection of the airway epithelium, the ASM itself constitutes an autologously regulated system that, when directly activated by RV, can also induce the mRNA expression and autocrine release and action of specific cytokines (notably, IL-1beta ), which lead to perturbations in ASM responsiveness. Thus, in agreement with previous studies on other resident stromal cells in the airways, such as fibroblasts and epithelial cells (7, 10, 30), our present observations are consistent with the notion that, under specific conditions (e.g., RV infection), ASM cells can also act as important contributors to tissue inflammation and intrinsically altered airway responsiveness.

Because our present observations in isolated ASM tissue and cultured ASM cells pertain to in vitro studies, the question is raised as to the relevance of these findings with respect to the in vivo state. Clearly, this fundamental question remains to be systematically addressed in appropriate in vivo studies, although our present observations provoke certain noteworthy considerations. Accordingly, in light of the evidence provided herein that implicates a role for direct RV infection of ASM in inducing its altered responsiveness, the compelling consideration is raised that RV-mediated changes in airway responsiveness in vivo may, at least in part, also be attributed to direct infection of the ASM by the viral pathogen. Such a process might involve extension of an initial RV infection of the airway epithelium into the adjacent ASM tissue. Alternatively, or possibly together with the latter mechanism, it is also conceivable that, in the presence of any RV-induced disruption of the integrity of the respiratory epithelium, infection by the virus of the underlying ASM may be facilitated. Another issue pertains to the potential role of ICAM-1 activation in vivo. In this connection, the present findings suggest that the effects of RV infection on ASM responsiveness are attributed to binding of the viral pathogen to its key endogenous receptor in ASM (i.e., ICAM-1). The latter also serves as the counterreceptor for the integrin lymphocyte function-associated antigen (LFA)-1, which is present on various leukocytes and is known to mediate a variety of cellular responses that are associated with altered local immune function. Given this evidence, the consideration is raised that, in addition to any potential direct coupling of RV to ICAM-I in ASM in the in vivo state, LFA-1-mediated activation of ICAM-1 in vivo may further serve to initiate and/or propagate the airway proinflammatory response to RV infection. The presence and relative contributions of the above potential in vivo mechanisms remain to be elucidated.

In conclusion, the present study examined the role and mechanism of action of RV infection of isolated ASM in mediating changes in agonist-induced ASM constrictor and relaxant responsiveness. The results demonstrated that 1) inoculation of ASM with RV induces enhanced ASM constrictor responsiveness to ACh and impaired beta -adrenoceptor-mediated ASM relaxation; 2) these changes in ASM responsiveness are mechanistically associated with RV-induced enhanced IL-1beta mRNA and protein expression, eliciting an autocrine action in ASM; and 3) the latter upregulated expression and autocrine action of IL-1beta is triggered by the binding of RV to its endogenous receptor, ICAM-1. Given the well-established clinical significance of RV infection in the pathogenesis of altered airway reactivity and asthma exacerbation, the above findings identify an important role and mechanism by which the ASM autologously induces its state of altered responsiveness after infection with RV.


    ACKNOWLEDGEMENTS

We thank J. Grunstein, S. Chang, and S. Ling for expert technical assistance and Margaret Brown for assistance with typing the manuscript.


    FOOTNOTES

This work was supported in part by the National Heart, Lung, and Blood Institute Grants HL-31467 and HL-58245, a Parker B. Francis Fellowship Award, and an Institutional Developmental Fund Award from the Joseph Stokes Jr. Research Institute of the Children's Hospital of Philadelphia.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. M. Grunstein, Division of Pulmonary Medicine, The Children's Hospital of Philadelphia, Univ. of Pennsylvania School of Medicine, 34th St. and Civic Center Blvd., Philadelphia, PA 19104.

Received 2 November 1998; accepted in final form 24 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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