Up to 80 percent of asthma exacerbations are associated with the presence of an upper respiratory viral infection (URI), with human rhinoviruses (HRV) being the viral type most commonly associated with asthma attacks. This proposal will test the hypothesis that virus induced production of the eosinophil chemotactic and activating cytokines, eotaxin, RANTES, and granulocyte-macrophage colony stimulating factor (GM-CSF) from infected epithelial cells plays a central role in enhancing bronchial eosinophilia, and that this increase in activated eosinophils in the airway triggers disease exacerbations in asthmatics. Since exogenously administered nitric oxide (NO) inhibits HRV replication and virally induced cytokine production, we suggest that the capacity to cause asthma exacerbations depends upon virus reaching the lower airways in sufficient amounts to override the inhibitory host NO response and trigger epithelial cytokine production. Moreover, eosinophils must be """"""""primed"""""""" to respond to the eosinophil chemoattractant cytokines. We suggest that this latter phenomenon occurs only in atopic individuals in response to circulating levels of priming cytokines (e.g. IL-5). To test these hypotheses, and to further delineate the role of NO in regulating HRV infections, we will use both in vitro studies of infection of respiratory epithelial cells and in vivo HRV infection of normal, atopic and asthmatic subjects. We will determine if NO inhibits HRV replication by enhancing host antiviral responses and/or by inhibiting the actions of the important HRV protease 2A and 3C. We will also determine if viral replication and/or cytokine production is inhibited by NO via inhibition of mitochondrial enzymes that are important in energy metabolism. We will use cells transfected with cDNA coding for inducible nitric oxide synthase (iNOS) to determine if endogenously produced NO has the same effects as exogenously administered NO on HRV replication and cytokine production. We will infect normal, atopic and asthmatic subjects with HRV in vivo and will relate the presence of HRV in lower airway epithelial cells to epithelial expression of mRNA and protein for eotaxin, RANTES, GM-CSF and iNOS. We will test the hypothesis that atopic subjects whose eosinophils, isolated just prior to infection, show transepithelial migration to RANTES/eotaxin in vitro, will show enhanced nasal and bronchial eosinophilia in vivo, and will determine if inhibition of iNOS in vivo enhances HRV induced epithelial cytokine production. These studies should provide new insights into the pathogenesis of viral exacerbations of asthma and may provide the basis for improved therapeutic interventions.
