RAPID COMMUNICATION
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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Human atopic asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and airway hyperresponsiveness. Recent studies demonstrate that the degree of airway responsiveness is strongly associated with interleukin (IL)-9 expression in murine lung. To investigate the contribution of IL-9 to airway hyperresponsiveness, and to explore directly its relationship to airway inflammation, we studied transgenic mice overexpressing IL-9. In this report we show that IL-9 transgenic mice (FVB/N-TG5), in comparison with FVB/NJ mice, display significantly enhanced eosinophilic airway inflammation, elevated serum total immunoglobulin E, and airway hyperresponsiveness following lung challenge with a natural antigen (Aspergillus fumigatus). These data support a central role for IL-9 in the complex pathogenesis of allergic inflammation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical and physiologic studies of asthma reveal an association with widespread narrowing of the airways and eosinophilic inflammation. The cells that infiltrate into the airways include not only eosinophils but also mast cells, immunoglobulin (Ig)E-producing plasma cells, and a specific subset of activated T cells referred to as Th2 lymphocytes (1, 2). Although the precise biologic mechanism underlying these cellular responses is unknown, it is clear that complex interactions between these cells in the airway mucosa and submucosa in response to antigen lead to the elaboration of cytokines, including interleukin (IL)-4, IL-5, and other inflammatory proteins producing chemical mediators that are critical in this disorder (2, 3). Despite these insights, a clear understanding of the pathogenesis of atopic asthma is lacking.
Significant biologic variability in airway responsiveness occurs in humans, and baseline airway hyperresponsiveness is recognized as a risk factor for asthma (4). Airway responsiveness also differs between inbred strains of mice and is strongly controlled by genetic factors (7). We have shown previously that airway hyperresponsiveness is mapped to a qualitative trait locus on mouse chromosome 13, where the IL-9 candidate gene is located (11). Moreover, differences between airway hyporesponsive C57BL/6J (B6) and hyperresponsive DBA/2J (D2) mice are associated with significant differences in lung IL-9 expression. Hyporesponsive B6 mice express undetectable levels of IL-9 protein at steady state, whereas hyperresponsive D2 mice have robust IL-9 protein levels in lung (11). Interestingly, the intermediate airway responsiveness of the (B6D2)F1 mice is associated with intermediate levels of IL-9 protein in lung. Thus, there appears to be a tight genotype-phenotype correlation.
A pleiotropic role for IL-9 in allergic asthma is suggested by several independent studies describing its functions in vitro and in vivo (12, 13). First, Th2 cells are well documented to have a crucial role in the development of the allergic response and are presumed to be associated with the pathogenesis of asthma (14, 15). IL-9 is a T-cell growth factor, and experimental data have shown a correlation between IL-9 production and Th2-responses in vivo (12). Second, mediator release from mast cells by allergen has long been considered a critical initiating event in allergy. IL-9 was originally identified as a mast-cell growth factor, and appears to upregulate the expression of mast-cell proteases, including mouse mast-cell protease (mMCP)-1, mMCP-2, mMCP-4 (16), and granzyme B (17). Thus, IL-9 may serve a role in the proliferation and differentiation of mast cells. Third, elevated IgE levels are considered a hallmark of atopic allergy and a risk factor for asthma (18), and studies have shown that IL-9 potentiates the release of IgE from primed B cells (19, 20). Fourth, IL-9 may affect IgE-mediated responses by upregulating the expression of the alpha chain of the high-affinity IgE receptor (17).
To investigate the contribution of IL-9 to airway hyperresponsiveness, and to explore directly its relationship to airway inflammation, we studied transgenic mice overexpressing IL-9. In this report we demonstrate that the degree of eosinophilic bronchial inflammation elicited using an antigen-challenge model of Aspergillus fumigatus (Af ) is significantly greater in the transgenic mice than in background control mice. Importantly, the enhanced inflammatory response to antigen produced by the overexpression of IL-9 is also associated with airway hyperresponsiveness. These data support a central role for the IL-9 pathway in the complex pathogenesis of allergic inflammation and asthma.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice
The following studies conformed to the principles for laboratory animal research outlined by the Animal Welfare
Act and the DHEW (National Institutes of Health, Washington, DC) guidelines for the experimental use of animals, and were approved by the Institutional Animal Care
and Use Committee. IL-9 transgenic mice were generated
in a FVB/N background using a fusion gene consisting of
an IL-9 genomic fragment linked to the promoter of the
murine pim-1 gene, including the TATA box and the cap
site, followed by two copies of the Eµ enhancer and one
copy of the mMLV long terminal repeats, as described
previously (21). FVB/NJ mice obtained from Jackson Laboratories (Bar Harbor, ME) were used as a genetic control
(transgene-negative). The IL-9 transgenic (FVB/N-TG5)
mice had serum levels of IL-9 
1 µg/ml in contrast to the
background strain (FVB/NJ), which had no detectable serum IL-9 (data not shown). The animals were housed in non-SPF conditions. The animal housing facilities were maintained at 22°C (range 19 to 24°C) with a light cycle of 12:12 h
light:dark. Food and water were provided ad libitum.
Antigen Sensitization and Challenge
Antigen sensitization and challenge were carried out essentially as described previously (22, 23). Mice were anesthetized by halothane inhalation, and 25 µl of Af (Bayer Pharmaceuticals, Elkhart, IN) extract antigen suspension (final dilution 1:50, wt/vol in 0.9% NaCl and 10% glycerol; n = 15 to 19 per strain) or vehicle (10% glycerol in 0.9% NaCl; n = 11 to 12 per strain) was applied to the left nare or mice were untreated (naive, n = 9 to 11 per strain). Animals were challenged three times a week for 3 wk with antigen, and were phenotyped approximately 12 h after the last exposure.
Pulmonary Functions
To determine the bronchoconstrictor response, respiratory system pressure was measured via a port in the tracheal cannula and continuously recorded before and during exposure to a bronchoconstrictor approximately 12 h after the last immunization. Mice were anesthetized and instrumented as previously described (7). The bronchoconstrictor response to 58 µg/kg of 5-hydroxytryptamine (5-HT; Sigma, St. Louis, MO), which is approximately midway in the dose-response curve for the FVB/NJ strain, was assessed by the change in peak inspiratory pressure (Ppi) integrated over time (5 min after bronchoconstrictor). This parameter, termed the airway pressure time index (APTI), is a simple and repeatable measure of the change in Ppi and is highly correlated with respiratory system resistance and elastance following a bronchoconstrictor challenge (24).
Bronchoalveolar Lavage
After measurement of lung function parameters, lungs were lavaged with three 0.5-ml washes of 0.9% sterile saline (room temperature [RT]). The washes were combined, and the lavage fluid was centrifuged (2,800 × g for 10 min at RT with an Eppendorf microcentrifuge 5415C; Eppendorf, Hamburg, Germany); the cell pellet was resuspended in 1 ml of Dulbecco's phosphate-buffered saline (PBS, without calcium or magnesium). Cells were then counted with a hemocytometer. An aliquot of the cell suspension was incubated with phloxine B (1:32 dilution in PBS) at RT for 10 min. The cells were counted with a hemocytometer, and eosinophils were defined as cells positively staining with positive morphology. Differential cell counts of bronchoalveolar lavages (BALs) from a random subset of mice from each group were made from slide preparations (Cytospin 3; Shandon, Pittsburgh, PA) stained with Kwik-Diffs stains (Shandon). Cells were identified as macrophages, eosinophils, neutrophils, and lymphocytes by standard morphology using light microscopy, and at least 100 cells were counted under ×400 magnification. The absolute number and percentage of each cell type were then calculated.
Serum Igs
Serum antibody levels were determined as previously described (25). In brief, blood was obtained from venipuncture of the abdominal vena cava after pulmonary functions
were completed. Blood was centrifuged (11,400 × g for 10 min at RT with an Eppendorf microcentrifuge 5415C), and
serum was separated and stored at 
20°C until analysis.
Microtiter plates (Corning, Corning, NY) were coated
with the capture antibody, either rat antimouse IgE or
IgG3 (Pharmingen, San Diego, CA), at concentrations of 2 or 8 µg/ml, respectively, in coating buffer and incubated
overnight at 4°C. Plates were washed three times with PBS-Tween and rinsed with PBS. Plates were blocked by addition of 0.2 ml of 1% bovine serum albumin for 30 min at
37°C and then washed three times without incubation. Samples or standards were added and plates were incubated for
2 h at RT. Mouse IgE and IgG3 standards (Pharmingen)
were used to generate standard curves. Following incubation, plates were washed three times with PBS-Tween and
then rinsed with PBS. Bound IgE and IgG3 were detected
using biotinylated detecting antibodies (Pharmingen), 2 and
8 µg/ml, respectively, then incubating 1 h at RT and washing three times. The assay was developed by adding avidin
peroxidase in substrate buffer with H2O2 (Sigma). Plates
were read at 405 nm on a Dynatech MR-5000 plate reader (Dynatech, Chantilly, VA). Concentrations of Igs were calculated by log-linear transformation of the standard curve.
Histology
Lungs were fixed in 10% neutral buffered formalin. Five-micrometer hematoxylin and eosin-stained sections were prepared. In addition, a Congo red stain was used to confirm the presence of eosinophils in the inflammatory infiltrate (26). This stain renders eosinophils red-orange in a blue background. As previously described, a grading screen was developed to characterize the intensity of the inflammatory infiltrate in lungs treated with vehicle or with Af antigen, or in naive lungs (22). Grade 0 lungs had very few scattered inflammatory cells, with no clustering of inflammatory cells around bronchi or vessels. Grade 1 lungs had one or two centrally located inflammatory cell aggregates. Grade 2 lungs had inflammatory aggregates located around a majority of bronchi and vessels in the central part of the lung. Grade 3 lungs showed extension of the inflammatory cuffs to the periphery of the lung approaching the pleura. Grading was performed blinded (by Dr. Matthijs van de Rijn) on slides that were not identified as to the treatment or the strain of mouse examined. Multiple sections were examined for each strain and condition.
Western Blots
Lung tissues were derived from various mice, dissected free of hilar lymph nodes, and snap-frozen in liquid nitrogen. Sections (27 mm3) were resuspended in 500 µl of 2× sodium dodecyl sulfate (SDS) lysis buffer (60 mM Tris, pH 6.8; 2% SDS; 0.1 M 2-mercaptoethanol; and 0.1% bromophenol blue) and boiled for 5 min. Twenty microliters of each lysate was electrophoresed on 18% tris-glycine SDS-polyacrylamide gel electrophoresis gels and electroblotted onto Immobilon-P (Millipore, Bedford, MA) membrane in transfer buffer (48 mM Tris, 40 mM glycine, 0.0375% SDS, and 20% methanol). Filters were blocked overnight in blocking buffer (Tris-buffered saline, 0.05% Tween-20, and 5% powdered milk). Filters were probed with polyclonal goat antimurine IL-9 (R&D Systems, Minneapolis, MN) and a secondary horseradish peroxidase- conjugated antigoat IgG (Pierce Chemical, Rockford, IL) and prepared for chemiluminescence. Filters were stained with Coomassie Brilliant Blue to assure equivalent amounts of protein.
Statistical Analyses
A two-way analysis of variance (ANOVA) with subsequent comparison by Bonferroni's t test was used for statistical analysis of APTI and eosinophil counts. Homogeneity of variances was tested using Levene's test, and data
with heterogeneous variances were transformed appropriately before applying ANOVA. Differences between means
were considered significant when yielding a P value 
0.05. Where data are presented as a percent change, the statistical analyses were conducted on the actual means and variances. Nonparametric analyses of BAL total cell count,
serum Igs, and lung histopathology scores were done by
means of Kruskal-Wallis test within strains with subsequent Dunn's tests, and Mann-Whitney rank sum test between strains. If between-strain or within-treatment data
had a normal distribution, a Student's t test was applied.
Differences between ranks were considered significant
when yielding a P value 0.05. Statistical analyses were
performed with the computer program SigmaStat (Jandel,
San Rafael, CA).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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IL-9 Overexpression in Lungs of Transgenic Mice
To investigate the contribution of IL-9 to airway responsiveness while controlling for genetic background, we studied FVB/NJ (transgene-negative control) and FVB/N-TG5 (IL-9 transgenic) mice. Steady-state IL-9 expression in the lungs of naive FVB/NJ and FVB/N-TG5 animals is shown in Figure 1. Using Western analyses, significantly greater IL-9 expression is seen in the lungs of FVB/N-TG5 mice than in lungs of FVB/NJ mice.
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Airway Hyperresponsiveness after Antigen in IL-9 Transgenic Mice
Measurements of airway responsiveness in IL-9 transgenic (FVB/N-TG5) animals and the background strain (FVB/ NJ) are shown in Figure 2. The APTI differed significantly between Af-challenged and vehicle-treated FVB/N-TG5 and FVB/NJ mice (P < 0.01). Although both strains demonstrated an increase in airway responsiveness after antigen, the APTI of Af-challenged FVB/N-TG5 mice was significantly greater than that of Af-challenged FVB/NJ mice (P < 0.05).
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BAL Cells Differ Significantly between Antigen-Challenged FVB/NJ and FVB/N-TG5 Mice
To explore further the role of IL-9 on airway inflammation, we measured BAL inflammatory cells in naive, vehicle-treated, and antigen-challenged FVB/N-TG5 and FVB/ NJ mice. BAL total cell counts and eosinophil counts did not differ statistically within or between naive strains (data not shown). Although eosinophil counts in vehicle-treated IL-9 transgenic mice were elevated compared with naive mice (P < 0.01), no significant elevation of eosinophils was noted in vehicle-treated FVB/NJ mice compared with naive animals (data not shown). BAL inflammatory cell counts differed significantly between antigen-challenged FVB/N-TG5 and FVB/NJ animals (5.3 ± 0.5 × 106 versus 1.8 ± 0.4 × 106 cells/lung, respectively, P < 0.01) (Table 1).
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Differential cell counts from the BALs of a subset of animals in each group demonstrated a predominance of eosinophils after antigen challenge in FVB/N-TG5 mice (Table 1). Importantly, total BAL eosinophil counts varied significantly between FVB/N-TG5 and FVB/NJ mice after animals were antigen challenged (P < 0.01) (data not shown).
Serum Total IgE Differs Significantly between Antigen-Challenged FVB/NJ and FVB/N-TG5 Mice
To investigate the role of IL-9 on serum Igs, we measured the serum total IgE and IgG3 in naive, vehicle-treated, and antigen-challenged FVB/N-TG5 and FVB/NJ mice. As a percent of control, serum total IgE levels in antigen-challenged FVB/N-TG5 mice (absolute value = 13,098 ± 2,898) were significantly greater (P < 0.01), than in antigen-challenged FVB/NJ mice (absolute value = 3,201 ± 675 ng/ml) (Figure 3, left). There was no significant difference in serum total IgE values within or between strains for either naive or vehicle-treated mice. Serum IgG3 was also monitored in these animals. No significant differences were observed in serum total IgG3 values within or between strains for naive, vehicle-treated, or antigen-challenged mice (Figure 3, right).
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significantly
different from FVB/NJ mice (same treatment) at P < 0.01 level.
There was no significant difference in serum total IgG3 within or
between strains for either naive, vehicle-treated, or antigen-treated mice. Number of antigen-treated mice is 18 per strain.
Lung Histopathology Differs Significantly between Antigen-Challenged FVB/NJ and FVB/N-TG5 Mice
To investigate further the role of IL-9 on the allergic inflammatory response, we carried out an anatomic and histologic examination of the lungs of a subset of naive and antigen-challenged FVB/N-TG5 and FVB/NJ mice. Histologic exam of a random subset of naive FVB/NJ mice (n = 2) showed no significant inflammatory infiltrate, and lungs were uniformly scored as Grade 0 (Figure 4A). The FVB/ NJ strain challenged with Af (n = 4) showed rare small inflammatory infiltrates and was scored Grade 1.125 (Figure 4B). Naive FVB/N-TG5 mice (n = 2) showed histology similar to the FVB/NJ mice, with rare scattered inflammatory cells in the lung parenchyma, and were uniformly scored as Grade 0 (Figure 4C). In contrast to the FVB/NJ strain, FVB/ N-TG5 mice (n = 5) after Af exposure showed a marked inflammatory infiltrate; the mean score for these animals was Grade 2.4. The inflammatory infiltrate in the Af-challenged FVB/N-TG5 mice showed dense cuffs of inflammatory cells surrounding bronchi (Figure 4E) and medium-sized vessels (Figure 4D). Occasionally, eosinophils were seen migrating through vessel walls (Figure 4D). The inflammatory infiltrate consisted of a mixture of eosinophils and lymphocytes. Only a few neutrophils were seen. Histiocytes were common in alveolar spaces but did not constitute a major component of the inflammatory cuffs themselves. In several areas eosinophils were seen to involve the bronchial epithelium, and the eosinophilic nature of the infiltrate was further highlighted by a Congo red stain (Figure 4F). All four groups differed significantly for lung histology grade (P = 0.01), and the histologic grade of the Af-challenged FVB/N-TG5 mice was significantly higher compared with Af-challenged FVB mice at the P < 0.05 level.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have previously identified the IL-9 gene as a candidate in allergic asthma on the basis of linkage homology between humans and mice for airway responsiveness (11, 25). To investigate the contribution of IL-9 to airway responsiveness and allergic inflammation while controlling for genetic background, we studied FVB/NJ and FVB/N-TG5 IL-9 transgenic mice. Consistent with our hypothesis, we showed that FVB/N-TG5 mice overexpressing IL-9 displayed many features pathognomonic of an asthmatic phenotype. First, these animals were relatively normal until lung exposure to a natural antigen, Af. This is not unlike humans, where asthma is best characterized by nonspecific intermittent symptoms, including shortness of breath, wheezing, coughing, and chest tightness, all correlated with bronchial inflammation related to antigen exposure (1, 2). Second, histologic and BAL examination confirmed that IL-9 promotes eosinophilic inflammation of the airways in vivo. In distinct contrast to the Af-challenged FVB/NJ control animals, the inflammatory response of Af-challenged FVB/N-TG5 mice was characterized by dense eosinophilic infiltrates accompanied by lymphocytes in peribronchial and perivascular areas extending deep into the lung. Moreover, eosinophils were also seen marginating and migrating through vessels walls, and were found in bronchial tissues and bronchiolar airspaces. These findings were corroborated by BAL investigations confirming markedly greater eosinophil counts in antigen-challenged FVB/N-TG5 mice when compared with FVB/NJ mice. Third, antigen exposure resulted in a significant increase in airway responsiveness in FVB/N-TG5 mice compared with FVB/NJ mice. Thus, airway hyperresponsiveness can be attributed to IL-9 overexpression after natural antigen exposure in this model system. In addition to these data, overexpression of IL-9 in these transgenic mice has been shown previously to be associated with intraepithelial mastocytosis, substantiating a role for IL-9 in mast-cell differentiation and proliferation in vivo (27).
Curiously, in the present report naive FVB/N-TG5 mice failed to display baseline airway hyperresponsiveness. One explanation is that the FVB/NJ and FVB/N-TG5 strains lack one or more additional factors necessary for the expression of baseline airway hyperresponsiveness. Nevertheless, these findings are not unlike previous IL-5 trangenics models (28, 29), in which systemic overexpression did not produce airway hyperresponsiveness. In contrast, IL-5 transgenic mice utilizing the Clara cell CC10 protein promoter did display baseline airway hyperresponsiveness (30). In the FVB/N-TG5 strain, Northern analyses demonstrate that IL-9 is expressed in all tissues, including lung epithelia (21). IL-9 was easily detected in the serum and and significantly increased in the whole lungs of FVB/N-TG5 mice at steady state, in contrast to the background control FVB/ NJ strain in which no IL-9 was detected in serum. Perhaps, in the absence of antigenic exposure, some threshold of lung IL-9 expression must be reached to produce baseline airway hyperresponsiveness. Perhaps the type and location of IL-9-producing cells are also important in producing baseline airway hyperresponsiveness in the case of IL-9, and a lung-specific promoter may be required.
We observed a significant difference between FVB/N-TG5 and FVB/NJ mice in serum total IgE response after antigen exposure. Although it is generally accepted that IL-4 is important in IgE-mediated responses, the role of IL-9 is less well understood. Whereas the precise mechanism by which IL-9 significantly enhances IgE production is unknown, in vitro studies have shown that IL-9 potentiates IL-4-induced IgE and IgG1 production from murine and human B lymphocytes (19, 20). Furthermore, we have demonstrated previously that recombinant IL-9 instillation into the lungs of the C57BL/6J strain, which is genetically deficient in IL-9, significantly increases serum total IgE production (25). In our studies only the IL-9 transgenic mice have significantly elevated serum total IgE levels, confirming that IL-9 is important in determining serum total IgE in vivo.
Based on current data, there is substantial support for IL-9 as a gene candidate in asthma. The pleiotropic role of IL-9 in vitro and in vivo described in independent studies (12, 13, 16, 17, 19, 20, 27, 31) supports a role for this cytokine in the allergic response. Moreover, the results of the current study, along with our recent data (11, 25), extend the known functions of this cytokine. The present in vivo data demonstrate a significant role for IL-9 in eosinophilic inflammation of the airways, and as a modulator of serum IgE and airway responsiveness, all significant risk factors for allergic asthma. These data suggest one of the broadest roles for IL-9 of any known cytokine associated with asthma, but additional work will be required to define a mechanism for these IL-9 activities. Further study of the IL-9 pathway, including the identification of associated genes and their functions, should provide additional useful insights into the pathogenesis of asthma. Moreover, novel therapies for allergic inflammatory disorders may be derived by blocking accessible targets in this pathway.
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Footnotes |
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Address correspondence to: Roy Clifford Levitt, M.D., Magainin Institute of Molecular Medicine, Magainin Pharmaceuticals, 5110 Campus Dr., Plymouth Meeting, PA 19462. E-mail: rlevitt{at}magainin.com
(Received in original form June 24, 1998 and in revised form August 10, 1998).
Note added in proof:Recently, genetic analysis of asthma kindreds has identified the human IL-9 receptor as a second asthma gene candidate (Grasso, L., M. Huang, C. D. Sullivan, C. J. Messler, M. B. Kiser, C. D. Dragwa, K. J. Holroyd, J.-C. Renauld, R. C. Levitt, and N. C. Nicolaides. 1998. Molecular analysis of human interleukin-9 receptor transcripts in peripheral blood mononuclear cells. J. Biol. Chem. 273:24016-24024). Analysis of IL-9 receptor transcripts has identified a nonfunctional receptor isoform that is variable among individuals (Holroyd, K. J., L. C. Martinati, E. Trabetti, T. Scherpbier, S. M. Eleff, A. L. Boner, P. F. Pignattti, M. B. Kiser, C. D. Dragwa, F. Hubbard, C. D. Sullivan, L. Grasso, C. J. Messler, M. Huang, Y. Hu, N. C. Nicolaides, K. H. Buetow, and R. C. Levitt. 1998. Asthma and bronchial hyperresponsiveness linked to the XY long arm pseudoautosomal region. Genomics 52:233-235). These data further support the data that IL-9 pathway is a mediator of the asthmatic response.
Acknowledgments: The authors thank Jamilia Louahed and Michael Zasloff for their thoughtful suggestions and advice, and Yaniv Tomer and Nancy Nixon for their expert technical assistance.
Abbreviations Af, Aspergillus fumigatus; ANOVA, analysis of variance; APTI, airway pressure time index; BAL, bronchoalveolar lavage; Ig, immunoglobulin; IL, interleukin; mMCP, mouse mast-cell protease; PBS, phosphate-buffered saline; RT, room temperature; SDS, sodium dodecyl sulfate.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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