Differential Proteomic Analysis of Bronchoalveolar Lavage Fluid in Asthmatics following Segmental Antigen Challenge

Abstract

Allergic asthma is characterized by persistent airway inflammation and remodeling. Bronchoalveolar lavage conducted with fiberoptic bronchoscopy has been widely used for investigating the pathogenesis of asthma and other lung disorders. Identification of proteins in the bronchoalveolar lavage fluid (BALF) and their expression changes at different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. In this report, we describe the first comprehensive differential proteomic analysis of BALF from both asthmatic patients and healthy subjects before and 24 h after segmental allergen challenge. Our proteomic analysis involves affinity depletion of six abundant BALF proteins, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS analysis in conjunction with database searching for protein identification and semiquantitation. More than 1,500 distinct proteins were identified of which about 10% displayed significant up-regulation specific to the asthmatic patients after segmental allergen challenge. The differentially expressed proteins represent a wide spectrum of functional classes such as chemokines, cytokines, proteases, complement factors, acute phase proteins, monocyte-specific granule proteins, and local matrix proteins, etc. The majority of these protein expression changes are closely associated with many aspects of the pathophysiology of asthma, including inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. Importantly a large portion of these proteins and their expression changes were identified for the first time from BALF, thus providing new insights for finding novel pathological mediators and biomarkers of asthma. Allergic asthma is characterized by persistent airway inflammation and remodeling. Bronchoalveolar lavage conducted with fiberoptic bronchoscopy has been widely used for investigating the pathogenesis of asthma and other lung disorders. Identification of proteins in the bronchoalveolar lavage fluid (BALF) and their expression changes at different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. In this report, we describe the first comprehensive differential proteomic analysis of BALF from both asthmatic patients and healthy subjects before and 24 h after segmental allergen challenge. Our proteomic analysis involves affinity depletion of six abundant BALF proteins, SDS-PAGE fractionation, protein in-gel digestion, and subsequent nano-LC-MS/MS analysis in conjunction with database searching for protein identification and semiquantitation. More than 1,500 distinct proteins were identified of which about 10% displayed significant up-regulation specific to the asthmatic patients after segmental allergen challenge. The differentially expressed proteins represent a wide spectrum of functional classes such as chemokines, cytokines, proteases, complement factors, acute phase proteins, monocyte-specific granule proteins, and local matrix proteins, etc. The majority of these protein expression changes are closely associated with many aspects of the pathophysiology of asthma, including inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. Importantly a large portion of these proteins and their expression changes were identified for the first time from BALF, thus providing new insights for finding novel pathological mediators and biomarkers of asthma. Asthma is a chronic lung disease characterized by reversible airway obstruction, inflammation, and bronchial hyperresponsiveness. The most common form of the disease, allergic asthma, results from inappropriate immune responses to common allergens in genetically susceptible individuals. After exposure to an allergen, such as ragweed or timothy grass, two phases of asthmatic responses can be distinguished (1Cohn L. Elias J.A. Chupp G.L. Asthma: mechanisms of disease persistence and progression.Annu. Rev. Immunol. 2004; 22: 789-815Google Scholar, 2Fehniger T.E. Sato-Folatre J.G. Malmstrom J. Berglund M. Lindberg C. Brange C. Lindberg H. Marko-Varga G. Exploring the context of the lung proteome within the airway mucosa following allergen challenge.J. Proteome. Res. 2004; 3: 307-320Google Scholar, 3Hamid Q. Tulic M.K. Liu M.C. Moqbel R. Inflammatory cells in asthma: mechanisms and implications for therapy.J. Allergy Clin. Immunol. 2003; 111: S5-S12Google Scholar, 4Persson C.G. Andersson M. Greiff L. Svensson C. Erjefalt J.S. Sundler F. Wollmer P. Alkner U. Erjefalt I. Gustafsson B. Airway permeability.Clin. Exp. Allergy. 1995; 25: 807-814Google Scholar, 5Pradalier A. Late-phase reaction in asthma: basic mechanisms.Int. Arch. Allergy Immunol. 1993; 101: 322-325Google Scholar). The early phase of bronchoconstriction reaction occurs within minutes of the allergen challenge and is initiated by smooth muscle contraction as a consequence of the activation and mediator release from local mast cells. A few hours after allergen exposure, a second and more severe response occurs that is accompanied by a long and increasing nonspecific bronchial airway hyperresponsiveness. This late stage response is characterized by the infiltration and accumulation of lymphocytes such as macrophages, neutrophils, and particularly eosinophils. of allergic asthma are many cells for different (1Cohn L. Elias J.A. Chupp G.L. Asthma: mechanisms of disease persistence and progression.Annu. Rev. Immunol. 2004; 22: 789-815Google Scholar, 3Hamid Q. Tulic M.K. Liu M.C. Moqbel R. Inflammatory cells in asthma: mechanisms and implications for therapy.J. Allergy Clin. Immunol. 2003; 111: S5-S12Google Scholar, 5Pradalier A. Late-phase reaction in asthma: basic mechanisms.Int. Arch. Allergy Immunol. 1993; 101: 322-325Google Scholar). The of mechanisms a of functional of different proteins involved in as as the specific disease Bronchoalveolar lavage used bronchoalveolar BALF, bronchoalveolar lavage segmental allergen in matrix tissue of that a in protein protein used bronchoalveolar BALF, bronchoalveolar lavage segmental allergen in matrix tissue of that a in protein protein conducted with fiberoptic bronchoscopy has been widely used to cells and other from the of fluid that the airway and the of bronchoscopy in asthma Scholar). Bronchoalveolar lavage fluid (BALF) thus different as as a wide of proteins that from the or are by and cells B. analysis of fluid and plasma in patients with acute lung J. 2004; Scholar, J.S. allergen challenge plasma protein into the of asthmatic subjects at hours at minutes after challenge.J. Clin. Scholar, A. C.G. A. M. Lindberg H. T.E. Marko-Varga G. bronchoalveolar analysis with 2003; 3: Scholar). to the of BALF proteins, analysis of BALF pathological mediators and more of many lung at the molecular In conjunction with segmental allergen challenge is a to the allergen challenge and responses in a in of bronchoscopy in asthma Scholar, A. C.G. A. M. Lindberg H. T.E. Marko-Varga G. bronchoalveolar analysis with 2003; 3: Scholar, G. P. of of fluid by lavage as of Scholar, I. A. P. 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J. in bronchoalveolar lavage fluid proteins in a J. 2003; Scholar, I. A. P. R. as the to for lung disease in bronchoalveolar Scholar, J. Malmstrom L. J. B. A. G. Marko-Varga G. and of the Res. 2004; 3: Scholar, R. J. M. I. proteome in a for 2003; 3: Scholar, J. and to lung J. 2004; Scholar, M. C. P. of protein A and changes of the bronchoalveolar lavage fluid proteome by in 22: Scholar, R. the of lung disease B. Scholar, R. P. of bronchoalveolar lavage B. Scholar). provide a more comprehensive BALF protein database and their in allergic asthma, we the first differential proteomic analysis of both from asthmatic patients and healthy subjects before and after segmental allergen challenge affinity depletion of six abundant proteins, SDS-PAGE of the proteins, and analysis for protein identification and semiquantitation. 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Tulic M.K. Liu M.C. Moqbel R. Inflammatory cells in asthma: mechanisms and implications for therapy.J. Allergy Clin. Immunol. 2003; 111: S5-S12Google A large of these proteins and their expression changes were identified for the first time from many of these to the pathophysiology of asthma. In this we a proteomic to the differential expression of proteins in In to that can or a few protein is to of proteins and thus comprehensive BALF proteome that is in the molecular mechanisms asthma. The of abundant proteins in BALF that of plasma proteins to the of In with the the most abundant plasma proteins such as and were to be most abundant in BALF from both asthmatic patients and healthy in for as as of the proteins in this BALF After the can further in BALF to plasma from into lung as a of damage to the J.S. allergen challenge plasma protein into the of asthmatic subjects at hours at minutes after challenge.J. Clin. Scholar). 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Immunol. 2004; Scholar). the to for and is a mediator that is to in the of H. in the of and Immunol. 2004; Scholar, J. G. H. to novel and for patients in the 2004; Scholar). The to other two and protein The pathophysiology of allergic asthma is complex and involves many (1Cohn L. Elias J.A. Chupp G.L. Asthma: mechanisms of disease persistence and progression.Annu. Rev. Immunol. 2004; 22: 789-815Google Scholar, 2Fehniger T.E. Sato-Folatre J.G. Malmstrom J. Berglund M. Lindberg C. Brange C. Lindberg H. Marko-Varga G. Exploring the context of the lung proteome within the airway mucosa following allergen challenge.J. Proteome. Res. 2004; 3: 307-320Google Scholar, 3Hamid Q. Tulic M.K. Liu M.C. Moqbel R. Inflammatory cells in asthma: mechanisms and implications for therapy.J. Allergy Clin. Immunol. 2003; 111: S5-S12Google Scholar, 5Pradalier A. Late-phase reaction in asthma: basic mechanisms.Int. Arch. Allergy Immunol. 1993; 101: 322-325Google Scholar, M. M. P. accumulation of in 2004; Scholar, L. of and in J. 2004; Scholar). The allergic response is by activation of mast cells. hours following allergen a of including macrophages, and are from the and the airway immune cells with the local smooth muscle and cells to the persistent airway inflammation and release of mediators such as chemokines, cytokines, and is an in the and expression of proteins within subjects with in in most of the differentially expressed proteins in can be to the different aspects of the complex molecular an asthmatic and cytokines, proteases, complement factors, acute phase proteins, and monocyte-specific granule proteins that are local matrix proteins that are associated with the mucus and of airway and abundant plasma proteins that the plasma infiltration. In the of protein expression changes with the of the response of an asthmatic to with and more protein with for and Our in BALF protein expression changes are with is about the pathophysiology of asthma. a and were identified in of the proteins and protein were to be after to were in with or and thus the in expression is is to that of the such as and and such as and protein were the of the BALF and tissue or and were the of and identified in in a new proteins associated with of inflammation the in proteases, complement factors, acute phase proteins, and and granule has been that activation of to the to of many granule proteins such as basic and etc. (1Cohn L. Elias J.A. Chupp G.L. Asthma: mechanisms of disease persistence and progression.Annu. Rev. Immunol. 2004; 22: 789-815Google Scholar, 3Hamid Q. Tulic M.K. Liu M.C. Moqbel R. Inflammatory cells in asthma: mechanisms and implications for therapy.J. Allergy Clin. Immunol. 2003; 111: S5-S12Google Scholar, M. M. P. accumulation of in 2004; Scholar). identified in with expression in after and responses to significant in the expression of these This by a that the of and their protein release in to the of the late phase response A. Late-phase reaction in asthma: basic mechanisms.Int. Arch. Allergy Immunol. 1993; 101: 322-325Google Scholar). the response of the asthmatic patients to is with the of a allergic asthma and thus further the as a for asthma In to the granule proteins, a of granule proteins, such as and were in the airway of asthmatic patients with the expression of proteins in healthy are or are to a following the molecular such changes in these proteins the of in the late phase reaction after are a large of that in asthma by their the and of cells and matrix and A. R. A. R. into the is a in a of Immunol. 2003; Scholar, A. release from Clin. Immunol. 2003; Scholar, R. H. in the pathogenesis of asthma and implications for 2004; 3: Scholar, in lung Res. Scholar, P. R. B. matrix in the airway after allergen J. Scholar). and were identified with in which with a in tissue and of to which is an of airway occurs A. R. A. R. into the is a in a of Immunol. 2003; Scholar, A. release from Clin. Immunol. 2003; Scholar, P. R. B. matrix in the airway after allergen J. Scholar). A in expression in after which the of in the of to be than that of A of proteins by local smooth muscle and in the airway were in BALF after of these proteins such as are involved in the of airway including protein and and are proteins that are of the in and airway a new protein J. 2004; Scholar, protein is in the airway response to Allergy Clin. Immunol. 2004; Scholar, M. The in and Rev. 2004; Scholar, J. M. 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More the which is a for the activation of the of in the BALF after This a response at the molecular to in the of BALF analysis in the pathogenesis of lung is to this the most comprehensive database of the proteins in of both healthy subjects and asthmatic This is the first of differential proteomic analysis of from both and healthy subjects before and after The analysis of and healthy in the identification of proteins which about 10% displayed expression for asthmatic patients at the of the late phase reaction to allergen challenge. the are a of asthmatic the from the protein the complex pathophysiology of asthma, such as inflammation, eosinophilia, airway remodeling, tissue damage and repair, mucus production, and plasma infiltration. This new insights for novel mediators that can be or biomarkers for asthma.