The Receptor for Advanced Glycation End Products Is a Central Mediator of Asthma Pathogenesis
2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.06.031
ISSN1525-2191
AutoresPavle S. Milutinovic, John F. Alcorn, Judson M. Englert, Lauren T. Crum, Tim D. Oury,
Tópico(s)Chronic Obstructive Pulmonary Disease (COPD) Research
ResumoThe receptor for advanced glycation end products (RAGE) is a multiligand receptor that has been shown to contribute to the pathogenesis of diabetes, atherosclerosis, and neurodegeneration. However, its role in asthma and allergic airway disease is largely unknown. These studies use a house dust mite (HDM) mouse model of asthma/allergic airway disease. Respiratory mechanics were assessed and compared between wild-type and RAGE knockout mice. Bronchovascular architecture was assessed with quantitative scoring, and expression of RAGE, immunoglobulins, and relevant cytokines was assessed by standard protein detection methods and/or quantitative RT-PCR. The absence of RAGE abolishes most assessed measures of pathology, including airway hypersensitivity (resistance, tissue damping, and elastance), eosinophilic inflammation, and airway remodeling. IL-4 secretion, isotype class switching, and antigen recognition are intact in the absence of RAGE. In contrast, normal increases in IL-5, IL-13, eotaxin, and eotaxin-2 production are abrogated in the RAGE knockouts. IL-17 indicates complex regulation, with elevated baseline expression in RAGE knockouts, but no induction in response to allergen. Treatment of WT mice with an inhibitor of RAGE markedly reduces inflammation in the HDM model, suggesting that RAGE inhibition may serve as a promising therapeutic strategy. Finally, the results in the HDM model are recapitulated in an ovalbumin model of asthma, suggesting that RAGE plays a role in asthma irrespective of the identity of the allergens involved. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that has been shown to contribute to the pathogenesis of diabetes, atherosclerosis, and neurodegeneration. However, its role in asthma and allergic airway disease is largely unknown. These studies use a house dust mite (HDM) mouse model of asthma/allergic airway disease. Respiratory mechanics were assessed and compared between wild-type and RAGE knockout mice. Bronchovascular architecture was assessed with quantitative scoring, and expression of RAGE, immunoglobulins, and relevant cytokines was assessed by standard protein detection methods and/or quantitative RT-PCR. The absence of RAGE abolishes most assessed measures of pathology, including airway hypersensitivity (resistance, tissue damping, and elastance), eosinophilic inflammation, and airway remodeling. IL-4 secretion, isotype class switching, and antigen recognition are intact in the absence of RAGE. In contrast, normal increases in IL-5, IL-13, eotaxin, and eotaxin-2 production are abrogated in the RAGE knockouts. IL-17 indicates complex regulation, with elevated baseline expression in RAGE knockouts, but no induction in response to allergen. Treatment of WT mice with an inhibitor of RAGE markedly reduces inflammation in the HDM model, suggesting that RAGE inhibition may serve as a promising therapeutic strategy. Finally, the results in the HDM model are recapitulated in an ovalbumin model of asthma, suggesting that RAGE plays a role in asthma irrespective of the identity of the allergens involved. The receptor for advanced glycation end products (RAGE) is a multiligand receptor first identified as a potential mediator in diabetes.1Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.J Biol Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar RAGE has many additional ligands, including S100 proteins, HMGB1, amyloid β, and heparin.2Hanford L.E. Enghild J.J. Valnickova Z. Petersen S.V. Schaefer L.M. Schaefer T.M. Reinhart T.A. Oury T.D. Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE).J Biol Chem. 2004; 279: 50019-50024Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 3Sparvero L.J. Asafu-Adjei D. Kang R. Tang D. Amin N. Im J. Rutledge R. Lin B. Amoscato A.A. Zeh H.J. Lotze M.T. RAGE (Receptor for Advanced Glycation Endproducts), RAGE ligands, and their role in cancer and inflammation.J Transl Med. 2009; 7: 17Crossref PubMed Scopus (461) Google Scholar The current paradigm maintains that membrane RAGE (mRAGE) signaling is proinflammatory, whereas soluble RAGE (sRAGE), a secreted form of RAGE, is generally anti-inflammatory because it scavenges proinflammatory ligands.4Ramasamy R. Yan S.F. Schmidt A.M. RAGE: therapeutic target and biomarker of the inflammatory response–the evidence mounts.J Leukoc Biol. 2009; 86: 505-512Crossref PubMed Scopus (236) Google Scholar RAGE transcript and protein are predominantly expressed in the lung,5Brett J. Schmidt A.M. Yan S.D. Zou Y.S. Weidman E. Pinsky D. Nowygrod R. Neeper M. Przysiecki C. Shaw A. et al.Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues.Am J Pathol. 1993; 143: 1699-1712PubMed Google Scholar and specifically by pulmonary type I alveolar epithelial cells,6Englert J.M. Hanford L.E. Kaminski N. Tobolewski J.M. Tan R.J. Fattman C.L. Ramsgaard L. Richards T.J. Loutaev I. Nawroth P.P. Kasper M. Bierhaus A. Oury T.D. A role for the receptor for advanced glycation end products in idiopathic pulmonary fibrosis.Am J Pathol. 2008; 172: 583-591Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar suggesting that RAGE has an important role in lung pathophysiology. Recent studies suggest that RAGE contributes to pulmonary disease; RAGE knockout mice are protected against hyperoxia-induced lung injury7Reynolds P.R. Schmitt R.E. Kasteler S.D. Sturrock A. Sanders K. Bierhaus A. Nawroth P.P. Paine III, R. Hoidal J.R. Receptors for advanced glycation end-products targeting protect against hyperoxia-induced lung injury in mice.Am J Respir Cell Mol Biol. 2010; 42: 545-551Crossref PubMed Scopus (69) Google Scholar and exhibit attenuated responses to bacterial pneumonia.8Ramsgaard L. Englert J.M. Manni M.L. Milutinovic P.S. Gefter J. Tobolewski J. Crum L. Coudriet G.M. Piganelli J. Zamora R. Vodovotz Y. Enghild J.J. Oury T.D. Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice.PLoS One. 2011; 6: e20132Crossref PubMed Scopus (44) Google Scholar RAGE also appears to have an important role in pulmonary fibrosis, but this depends on the type of injury model used to generate the fibrotic response.6Englert J.M. Hanford L.E. Kaminski N. Tobolewski J.M. Tan R.J. Fattman C.L. Ramsgaard L. Richards T.J. Loutaev I. Nawroth P.P. Kasper M. Bierhaus A. Oury T.D. A role for the receptor for advanced glycation end products in idiopathic pulmonary fibrosis.Am J Pathol. 2008; 172: 583-591Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 9Englert J.M. Kliment C.R. Ramsgaard L. Milutinovic P.S. Crum L. Tobolewski J.M. Oury T.D. Paradoxical function for the receptor for advanced glycation end products in mouse models of pulmonary fibrosis.Int J Clin Exp Pathol. 2011; 4: 241-254PubMed Google Scholar, 10He M. Kubo H. Ishizawa K. Hegab A.E. Yamamoto Y. Yamamoto H. Yamaya M. The role of the receptor for advanced glycation end-products in lung fibrosis.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L1427-L1436Crossref PubMed Scopus (135) Google Scholar, 11Ramsgaard L. Englert J.M. Tobolewski J. Tomai L. Fattman C.L. Leme A.S. Kaynar A.M. Shapiro S.D. Enghild J.J. Oury T.D. The role of the receptor for advanced glycation end-products in a murine model of silicosis.PLoS One. 2010; 5: e9604Crossref PubMed Scopus (30) Google Scholar RAGE expression is altered in these models of disease, for example, the appearance of sRAGE in the bronchoalveolar lavage fluid (BALF) in a mouse model of pneumonia and loss of RAGE expression in models of pulmonary fibrosis. Asthma/allergic airway disease (AAD) is a main inflammatory condition of modern industrial societies, seen with increasing frequency throughout the developing world. Bronchodilators and corticosteroids remain the mainstays of therapy, but they are ineffective or inadequate for some groups of patients. Novel therapies that exploit auxiliary mechanisms of disease and that incur fewer side effects than chronic corticosteroid treatment are greatly needed. To date, there appears to have been no studies of RAGE in animal models of asthma. A few studies in humans have suggested that there is an increase in the levels of RAGE ligands HMGB112Watanabe T. Asai K. Fujimoto H. Tanaka H. Kanazawa H. Hirata K. Increased levels of HMGB-1 and endogenous secretory RAGE in induced sputum from asthmatic patients.Respir Med. 2011; 105: 519-525Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar and S100A8/A913Halayko A.J. Ghavami S. S100A8/A9: a mediator of severe asthma pathogenesis and morbidity?.Can J Physiol Pharmacol. 2009; 87: 743-755Crossref PubMed Scopus (67) Google Scholar in samples from patients with asthma compared with controls, suggesting that RAGE may contribute to asthma/AAD pathogenesis. Although one recent study suggested that sRAGE is increased concomitantly with HMGB1 in patients with asthma,12Watanabe T. Asai K. Fujimoto H. Tanaka H. Kanazawa H. Hirata K. Increased levels of HMGB-1 and endogenous secretory RAGE in induced sputum from asthmatic patients.Respir Med. 2011; 105: 519-525Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar another suggested a decrease in sRAGE and no change in HMGB1 in patients with neutrophilic asthma.14Sukkar M.B. Wood L.G. Tooze M. Simpson J.L. McDonald V.M. Gibson P.G. Wark P.A. Soluble RAGE is deficient in neutrophilic asthma and chronic obstructive pulmonary disease.Eur Respir J. 2012; 39: 721-729Crossref PubMed Scopus (108) Google Scholar Apart from the potential inconsistency between the latter two results, those studies have not provided mechanistic insight as to the role of mRAGE versus sRAGE in asthma, nor have they elucidated how cytokines and chemokines key to allergic disease are differentially regulated in the presence or absence of RAGE. The present study used a house dust mite (HDM) antigen sensitization/challenge model of asthma/AAD to directly assess the role of RAGE in asthma/AAD. An advantage of this model is that sensitization and challenge are effected by intranasal HDM extract application in the absence of adjuvant. Moreover, HDM antigen has been identified as a key contributor to asthma pathogenesis in humans and is a known trigger of acute asthmatic exacerbations.15Gavett S.H. Koren H.S. The role of particulate matter in exacerbation of atopic asthma.Int Arch Allergy Immunol. 2001; 124: 109-112Crossref PubMed Scopus (78) Google Scholar, 16Thomas W.R. Hales B.J. Smith W.A. House dust mite allergens in asthma and allergy.Trends Mol Med. 2010; 16: 321-328Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar To strengthen the applicability of the results and to further clarify mechanism, analogous experiments were also performed in an ovalbumin model of AAD to ensure that any effects of RAGE are not specific to the model investigated. Wild-type (WT) C57BL/6 strain mice subjected to these models develop airway hyperresponsiveness to methacholine challenge; bronchial, vascular, and interstitial eosinophilia; goblet cell hyperplasia with mucus hypersecretion; and elevated titers of IgE, the immunoglobulin class most closely associated with allergic disease. WT male C57BL/6 mice were purchased from Taconic (Hudson, NY). Founder RAGE knockout (RAGE KO) mice were provided by Dr. A. Bierhaus (University of Heidelberg), and from these mice a breeding colony was initiated.6Englert J.M. Hanford L.E. Kaminski N. Tobolewski J.M. Tan R.J. Fattman C.L. Ramsgaard L. Richards T.J. Loutaev I. Nawroth P.P. Kasper M. Bierhaus A. Oury T.D. A role for the receptor for advanced glycation end products in idiopathic pulmonary fibrosis.Am J Pathol. 2008; 172: 583-591Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 11Ramsgaard L. Englert J.M. Tobolewski J. Tomai L. Fattman C.L. Leme A.S. Kaynar A.M. Shapiro S.D. Enghild J.J. Oury T.D. The role of the receptor for advanced glycation end-products in a murine model of silicosis.PLoS One. 2010; 5: e9604Crossref PubMed Scopus (30) Google Scholar, 17Constien R. Forde A. Liliensiek B. Grone H.J. Nawroth P. Hammerling G. Arnold B. Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line.Genesis. 2001; 30: 36-44Crossref PubMed Scopus (233) Google Scholar These mice are congenic with the C57BL/6 background. RAGE KO mice were age- and sex-matched to WT mice for each experiment. In all cases mice were housed in the animal care facility of the University of Pittsburgh, and experimental protocols were approved by the University's Institutional Animal Care and Use Committee. HDM extract was obtained from Greer Laboratories (Lenoir, NC). Mouse serum albumin (MSA) was purchased from Sigma (St. Louis, MO). sRAGE was purified from mouse lung tissue, and endotoxin was removed with a Detoxi-Gel column (Thermo-Fisher) as described previously.2Hanford L.E. Enghild J.J. Valnickova Z. Petersen S.V. Schaefer L.M. Schaefer T.M. Reinhart T.A. Oury T.D. Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE).J Biol Chem. 2004; 279: 50019-50024Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 18Englert J.M. Ramsgaard L. Valnickova Z. Enghild J.J. Oury T.D. Large scale isolation and purification of soluble RAGE from lung tissue.Protein Expr Purif. 2008; 61: 99-101Crossref PubMed Scopus (10) Google Scholar Purity was confirmed by SDS-PAGE and Coomassie Brilliant Blue staining. Specific binding of the purified sRAGE to a known RAGE ligand, HMGB1, was assessed as described elsewhere19Demling N. Ehrhardt C. Kasper M. Laue M. Knels L. Rieber E.P. Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells.Cell Tissue Res. 2006; 323: 475-488Crossref PubMed Scopus (189) Google Scholar to ensure ligand binding was intact in the purified protein. AAD/asthma was induced in mice with the use of one of three protocols. In the first protocol, 8-week-old male WT or RAGE KO mice were treated intranasally (i.n.) four times per week for 7 weeks with 40 μg of HDM extract in 25 μL of saline. Control mice were treated with saline vehicle alone. Mice were sacrificed 48 hours after the last treatment. In the second protocol, 8-week-old male WT C57BL/6 mice were treated i.n. five times per week for 3 weeks with one of the following six treatments [in each case in 25 μL of phosphate-buffered saline (PBS)]: saline control, 25 μg of MSA alone, 25 μg of mouse sRAGE alone, 40 μg of HDM extract alone, 40 μg of HDM extract with 25 μg of MSA, or 40 μg of HDM extract with 25 μg of sRAGE. The third protocol used ovalbumin as the sensitization/challenge antigen. Sensitization was effected by intraperitoneal treatment with 50 μg of ovalbumin (Sigma) with 2 mg of aluminum hydroxide gel (Brenntag, Reading, PA) in 0.5 mL of saline on days 0 and 7. Control mice were given alum with saline alone. Intranasal challenge commenced at day 14 with 10 μg of ovalbumin in 25 μL of saline (or saline alone, in controls), continuing on alternate days for a total of three treatments per week for 3 weeks. Mice were sacrificed 24 hours after the last treatment. Pulmonary function was assayed with a flexiVent apparatus as described elsewhere.20Alcorn J.F. Rinaldi L.M. Jaffe E.F. van Loon M. Bates J.H. Janssen-Heininger Y.M. Irvin C.G. Transforming growth factor-beta1 suppresses airway hyperresponsiveness in allergic airway disease.Am J Respir Crit Care Med. 2007; 176: 974-982Crossref PubMed Scopus (103) Google Scholar Briefly, mice were anesthetized with pentobarbital, underwent tracheotomy and cannula placement, and were coupled to the flexiVent ventilator apparatus (SCIREQ, Montreal, QC). Mice were ventilated with a 0.2 mL tidal volume and positive end-expiratory pressure of 3 cm of H2O. Pressure and volume were measured and were fit by multiple linear regression to a linear model of the lung. Methacholine was delivered via a nebulizer; after each dose, the response was measured by applying 2-second perturbations at 10-second intervals for a total of 3 minutes. Dose response curves were then determined for each of three parameters that measured lung function. After pulmonary function testing, the mice were exsanguinated, and sera were prepared with the use of serum separator tubes (Becton Dickinson, Franklin Lakes, NJ). Saline (0.8 mL) was instilled in the lungs via the trachea and withdrawn. BALF cell counts were obtained, and cytospin slides were prepared for differential cell counting. Slides were stained with Diff-Quik (Siemens, Washington, DC) and air dried, and Permount coverslips (Fisher, Pittsburgh, PA) were placed on the slides. The relative percentages of monocytes, eosinophils, neutrophils, and lymphocytes were determined by counting five high-power fields. After centrifugation, BALF supernatant fluid and sera were frozen at −80°C for future studies. RNA was prepared from whole lung with the use of an RNeasy Mini Kit (Qiagen, Valencia, CA), per the manufacturer's instructions. Reverse transcription was performed with Moloney murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) in a Techne thermal cycler (Bibby Scientific US, Burlington, NJ). Quantitative RT-PCR (qRT-PCR) was performed with universal PCR buffer and TaqMan primer/probe assay reagent (Applied Biosystems) with primers for RAGE (Mm00545815 m1), IL-5 (Mm00439646 m1), IL-13 (Mm00505403 m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control (Mm99999915 g1). The following sequence was performed on an ABI Prism 7300 machine (Applied Biosystems): 50°C (2 minutes), 95°C (10 minutes), and then 40 cycles of 95°C (15 seconds) followed by 60°C (1 minute). The fold change in RAGE, IL-5, or IL-13 mRNA expression, compared with GAPDH mRNA housekeeping control, was determined with the ΔΔCt method. Lung homogenates were prepared by homogenizing and sonicating mouse lungs in cold CHAPS (3[lsqb[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid) buffer with protease inhibitors (150 mmol/L NaCl, 50 mmol/L Tris-HCl, 10 mmol/L CHAPS, 100 μmol/L 3,4-dichloroisocoumarin, 10 μmol/L E-64, 2 mmol/L ortho-phenanthroline, pH 7.4), followed by protein extraction over several hours at 4°C. Total protein content in lung homogenates was determined with the Bradford method. Ten micrograms of lung homogenate protein, 65 μL of undiluted BALF, or 65 μL of 1:10 diluted (in PBS) serum samples, were analyzed by SDS-PAGE and immunoblotting as described previously.21Bury A.F. Analysis of protein and peptide mixtures - evaluation of 3 sodium dodecyl sulfate-polyacrylamide gel-electrophoresis buffer systems.J Chromatogr. 1981; 213: 491-500Crossref Scopus (203) Google Scholar After blocking in 5% milk, membranes were incubated in 1:5000 primary rabbit anti-RAGE polyclonal antibody (GenScript, Piscataway, NJ) followed by 1:5000 secondary horseradish peroxidase anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA). For studies that explored the antigen binding profile of serum IgG from sensitized and challenged mice, 100 μg of HDM extract was separated by SDS-PAGE under reducing (50 mmol/L dithiothreitol) and nonreducing (no dithiothreitol) conditions, then transferred to membranes and blocked in 5% milk. Membranes were then incubated in 1:20 diluted pooled sera from sensitized and challenged WT or RAGE KO mice followed by 1:5000 secondary horseradish peroxidase anti-mouse IgG antibody (Jackson ImmunoResearch). Membranes were developed with enhanced chemiluminescent detection (Thermo-Fisher, Waltham, MA). Loading control was performed with Ponceau S staining for BALF and serum and by stripping and reprobing with an antibody against β-actin (Sigma) on lung homogenate blots. Paraffin-embedded inflation-fixed lungs were sectioned at a 5-μm thickness, mounted on SuperFrost Plus slides (Fisher), air dried, melted, deparaffinized with xylenes, and rehydrated with graded ethanol solutions. Antigen retrieval was performed in 0.2 N HCl with 1 mg/mL pepsin (Fisher) at 37°C for 10 minutes. Between each step, three 5-minute washes with PBS or PBS containing Tween-20 (PBST) were performed. Sections were blocked with 2% bovine serum albumin in PBST, whereas primary and secondary antibody treatments were performed in 0.5% bovine serum albumin in PBST. To detect RAGE, 1:500 diluted goat polyclonal antibody in serum raised against full-length mouse sRAGE (GenScript) was used, followed by 1:500 diluted donkey anti-goat antibody conjugated to cyanine 3 (Jackson ImmunoResearch). Control preimmune serum from the same animal in which the RAGE antibody was raised and additionally RAGE KO lung sections were used to confirm specificity. Nuclei were stained with a brief application of Hoechst stain (10 mg/mL). Sections were coverslipped with gelvatol and examined by an Olympus IX71 inverted microscope (Olympus, Tokyo, Japan). All images were processed with ImageJ software version 1.44o (NIH, Bethesda, MD).22Abramoff M.D. Magalhaes P.J. Ram S.J. Image processing with ImageJ.Biophotonics Int. 2004; 11: 36-42Google Scholar Images were background subtracted as individual colors, followed by color channels being merged. In all cases a rolling ball radius of 50.0 pixels was used. Lungs were inflation fixed with 10% neutral-buffered formalin, paraffin embedded, cut into sections 5 μm thick, stained with H&E or PAS (Research Histology Services, University of Pittsburgh), and examined by light microscopy All images were processed with ImageJ software version 1.44o. Images were light background subtracted. In all cases a rolling ball radius of 50.0 pixels was used. Eotaxin, eotaxin-2 (both from Abcam, Cambridge, MA), IL-4, IL-5 (both from BD Biosciences, San Jose, CA), IL-17 (R&D Systems, Minneapolis, MN), and IL-13 (eBioscience, San Diego, CA) enzyme-linked immunosorbent assays (ELISAs) were performed per manufacturer's instructions, using neat or diluted BALF in all cases except for IL-17 ELISA, which used neat lung homogenate. HDM-specific IgG1 ELISA was performed by coating absorbent plates with 2 μg/mL HDM extract in coating buffer (100 mmol/L NaHCO3, 30 mmol/L Na2CO3, pH 9.5) overnight at 4°C. Between each step, washes were performed with PBST. After blocking with 1% bovine serum albumin, 1:100 diluted serum or neat lung homogenate (in CHAPS buffer) was applied and incubated at 4°C overnight with agitation. Detection was performed by treating with 1:1000 diluted goat anti-mouse IgG1 conjugated to horseradish peroxidase (Jackson ImmunoResearch), and then o-phenylenediamine dihydrochloride (OPD) substrate (Sigma). Plates were read at 450 nm, and data were recorded as absolute absorbances. Total IgE ELISA was performed with an OptEIA IgE ELISA kit (BD Biosciences) per the manufacturer's instructions. Serum samples were diluted 1:20 in assay diluent. For HDM-specific ELISA, HDM extract was first biotinylated with the use of 6-((biotinoyl)amino)hexanoic acid, sulfosuccinimidyl ester (biotin-X, SSE) (Invitrogen, Carlsbad, CA). Then, free biotin was removed with a Sephadex G-25 quick spin column (Roche, Indianapolis, IN). Plates were incubated overnight at 4°C with capture antibody (OptEIA IgE ELISA kit), then application of 1:5 diluted (in assay diluent) serum overnight at 4°C with agitation. Biotinylated HDM extract was applied to detect HDM-specific IgE. This was followed by streptavidin conjugated to horseradish peroxidase (OptEIA IgE ELISA kit), and then OPD substrate. The plates were read at 450 nm, and data were recorded as absolute absorbances. H&E-stained lung sections from C57BL/6 mice were examined by a board-certified pathologist (T.D.O.) who had been blinded to the identity of the samples. The total number of bronchovascular bundles in each lung section was counted, and inflammation was scored. Inflammation (if any) in each bronchovascular bundle was graded as 0 (none), 1 (mild), 2 (moderate), or 3 (severe). The results are expressed as the percentage of all bronchovascular bundles that involve any inflammatory infiltrates, and, in addition, as an average severity score obtainable by dividing the pan-section sum of the bronchovascular bundle inflammation scores by the total number of bronchovascular bundles in that section. Statistical analysis was performed with GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA), and quantitative results were expressed as means ± SEMs. Statistical significance was determined with two-way analysis of variance and, when appropriate, unpaired Student's t-test. A P value < 0.05 was considered to be significant. To investigate whether RAGE expression was altered in mice sensitized and challenged with HDM extract compared with vehicle-treated controls, qRT-PCR and immunoblotting on whole lung homogenates were performed. Although there was a modest decrease in RAGE transcript expression in the lungs of mice treated with HDM extract compared with saline-treated controls (Figure 1A), this difference was not statistically significant. No change was observed in the overall expression level of RAGE protein or in the relative proportions of sRAGE versus two different isoforms of membrane-bound RAGE, xRAGE,23Gefter J.V. Shaufl A.L. Fink M.P. Delude R.L. Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines.Cell Tissue Res. 2009; 337: 79-89Crossref PubMed Scopus (27) Google Scholar and mRAGE (Figure 1B). Finally, sRAGE could not be detected in BALF or serum of either sensitized/challenged or naive mice (Figure 1C). Because RAGE is abundantly expressed by type I alveolar epithelial cells, it was thought that subtle changes in RAGE expression in other cell types more closely associated with asthma/AAD, such as bronchial epithelial cells or inflammatory cells, might be undetectable by whole lung expression assays. Immunofluorescence microscopy studies of lung sections performed to address this question showed no appreciable change in bronchial or vascular RAGE expression in response to HDM extract sensitization and challenge, and expression in type I alveolar epithelial cells appeared unaltered (Figure 1D). Expression of a pathogenetic mediator need not be altered in a disease state, particularly when the mediator is a receptor with multiple, potentially mutually antagonistic, roles. Therefore, to explore whether the absence of RAGE dampens or augments asthma/AAD, RAGE KO mice on the C57BL/6 background were subjected to the HDM extract sensitization/challenge protocol, alongside WT mice for comparison. Pulmonary function testing showed significant alterations in airway function in WT mice treated with HDM extract, consistent with an asthmatic profile (Figure 2A). These changes in responsiveness to methacholine challenge were evident in the parameters corresponding to large airway resistance (Rn), small airway tissue damping (G), and tissue elastance (H), respectively. The modest change in Rn, compared with what is often seen in studies in BALB/c mice, was consistent with findings of previous studies in the C57BL/6 strain.24Alcorn J.F. Ckless K. Brown A.L. Guala A.S. Kolls J.K. Poynter M.E. Irvin C.G. van der Vliet A. Janssen-Heininger Y.M. Strain-dependent activation of NF-kappaB in the airway epithelium and its role in allergic airway inflammation.Am J Physiol Lung Cell Mol Physiol. 2010; 298: L57-L66Crossref PubMed Scopus (19) Google Scholar The picture with the Rn parameter was somewhat complex, with a slightly elevated responsiveness to methacholine in naive RAGE KO mice compared with WT counterparts, but no difference between saline- versus HDM extract-treated RAGE KO mice was seen. Because there were no observable differences in airway or parenchymal architecture in RAGE KO mice, it is likely that any intrinsic changes in methacholine responsiveness in this strain are due to a minor effect of RAGE (directly or by compensatory gene expression) on airway smooth muscle function (see Discussion for the potential role of IL-17). However, it is important to note that RAGE KO mice treated with HDM extract showed G and H parameters indistinguishable from those of naive WT and RAGE KO mice. Modified Romanowsky staining of BALF cells showed markedly elevated cell counts and eosinophilia in the allergic WT mice compared with controls (Figure 2B). Histologic evaluation of lung sections indicated peribronchial, perivascular, and interstitial eosinophilia in lungs of allergic WT mice by H&E staining (Figure 2C), whereas PAS staining showed goblet cell hyperplasia and elevated expression of mucin in numerous bronchi of the allergic WT mice compared with controls (Figure 2D). In contrast to WT mice, RAGE KO mice had essentially no inflammatory infiltrates and no elevated mucin expression or goblet cell hyperplasia. These results suggested that RAGE plays a vital role in the key tissue changes observed in asthma/AAD, including airway hyperresponsiveness, eosinophilic peribronchial infiltrates, mucus hypersecretion, and airway remodeling. To determine whether the humoral immune response to HDM extract sensitization and challenge was altered in RAGE KO mice, ELISA was performed on sera and lung homogenates of WT and RAGE KO mice to evaluate the levels of HDM-specific and total immunoglobulins. Interestingly, significantly elevated levels of HDM-specific IgG1 in serum and lung homogenate of both WT and RAGE KO mice that had been sensitized and challenged with HDM extract were observed (Figure 3A). To test whether there were broad differences between the HDM antigen binding profiles of serum IgG from allerge
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