UVM Theses and Dissertations
Format:
Print
Author:
McElhinney, Brian
Dept./Program:
Cell and Molecular Biology Program
Year:
2005
Degree:
Ph. D.
Abstract:
The connection between eosinophils and asthma has been studied for many years. In this time, we have learned a great deal about these leukocytes, as well as the pathogenesis of asthma. It is now relatively certain that eosinophils play some role in disease, yet we do not understand which processes they drive, nor do we understand why these cells exist. Eosinophils are unique among leukocytes in that they predominantly contain highly cationic proteins in their granules. These proteins are released from eosinophils through a process known as degranulation. Eosinophil granule proteins can be detected in the lungs of asthmatics, and the amount of degranulated eosinophil proteins in the lung has been shown to correlate with asthma severity. The initial focus of this thesis research was to study the effects of a purified eosinophil granule protein, eosinophil peroxidase (EPO), on lung epithelial cells in vitro. EPO has significant potential to induce cellular injury as it is both cationic and a peroxidase. The formation of 3-nitrotyrosine residues is a marker of peroxidase activity, and lung epithelial cells exposed to EPO in the presence of H₂0₂ and nitrite contained significant amounts of 3-nitrotyrosine. Cells exposed to EPO were found to strongly activate the protein c-Jun-NH₂-terminal kinase (JNK). Membrane blebbing is a characteristic response to both noxious and apoptotic stimuli, and treatment of cells with active EPO caused JNK-dependent membrane blebbing. Interestingly, inhibition of the Rho-Kinases (ROCK-1, ROCK-2) blocked the EPO induced membrane blebbing. These data were the first to show that cationic proteins activate JNK in cells and to suggest that Rho kinases may be upstream activators of JNK.
To better understand the importance of cationic proteins in asthma, we next examined the effects of cationic proteins in vivo. Intratracheal instillation of cationic proteins lead to the activation of JNK in the lung epithelium, and caused airway hyperresponsiveness (AHR). AHR is a characteristic phenotype of asthma where inhaled cholinergic agonists generate smooth muscle constriction, which is greater in asthmatics. Instillation of cationic proteins to mice lacking JNK-1 (JNK⁻/⁻) did not cause AHR. This unexpected finding led us to further explore the importance of JNK-1 in regulating AHR. The induction of AHR by antigens, as is the case in many asthmatics, requires the presence of eosinophils in mouse models. In a model of allergic airway inflammation, JNK-I⁻/⁻ mice have a hypereosinophilia, as compared to normals, yet do not develop AHR. JNK activity was detectable in several cell types in inflamed lungs of wild-type mice. Mice lacking eosinophils (Phil) failed to activate JNK in the lung beyond control levels in response to antigen. This data was the first to show direct dependence of AHR on JNK-1, and to demonstrate that eosinophils are significant JNK activators in allergic airway disease. In order to pursue the importance of JNK activation in the lung epithelium, we generated transgenic mice expressing a doxycycline inducible, dominant-negative form of JNK-1 in lung epithelial cells. Induction of the transgene lead to significant changes in lung structure, particularly in the airway epithelium and most strikingly in the alveoli, resembling microatelectasis. Transgene induction also caused minor patches of inflammation focused around airways and blood vessels and displayed evidence of lung remodeling. Thus, a baseline homeostatic level of JNK activity in the lung epithelium appears to be essential for normal lung function.
To better understand the importance of cationic proteins in asthma, we next examined the effects of cationic proteins in vivo. Intratracheal instillation of cationic proteins lead to the activation of JNK in the lung epithelium, and caused airway hyperresponsiveness (AHR). AHR is a characteristic phenotype of asthma where inhaled cholinergic agonists generate smooth muscle constriction, which is greater in asthmatics. Instillation of cationic proteins to mice lacking JNK-1 (JNK⁻/⁻) did not cause AHR. This unexpected finding led us to further explore the importance of JNK-1 in regulating AHR. The induction of AHR by antigens, as is the case in many asthmatics, requires the presence of eosinophils in mouse models. In a model of allergic airway inflammation, JNK-I⁻/⁻ mice have a hypereosinophilia, as compared to normals, yet do not develop AHR. JNK activity was detectable in several cell types in inflamed lungs of wild-type mice. Mice lacking eosinophils (Phil) failed to activate JNK in the lung beyond control levels in response to antigen. This data was the first to show direct dependence of AHR on JNK-1, and to demonstrate that eosinophils are significant JNK activators in allergic airway disease. In order to pursue the importance of JNK activation in the lung epithelium, we generated transgenic mice expressing a doxycycline inducible, dominant-negative form of JNK-1 in lung epithelial cells. Induction of the transgene lead to significant changes in lung structure, particularly in the airway epithelium and most strikingly in the alveoli, resembling microatelectasis. Transgene induction also caused minor patches of inflammation focused around airways and blood vessels and displayed evidence of lung remodeling. Thus, a baseline homeostatic level of JNK activity in the lung epithelium appears to be essential for normal lung function.