Viral infections are a major cause of asthma attacks. Neural control of the airways is markedly abnormal in both humans and experimental animals with viral airway infections. Under normal circumstances, the release of acetylcholine from airway vagal fibers is limited by inhibitory M2 muscarinic receptors on the nerve endings. Loss of these receptors during viral infections increases bronchoconstriction. Host cells express interferons (IFNs) in response to viral infection. We have demonstrated that IFN-gamma causes M2 receptor dysfunction in cultured airway parasympathetic neurons. IFN-beta, as well as double-stranded RNA (a potent stimulus to IFN-alpha and beta production during viral infections) cause M2 receptor dysfunction and airway hyperreactivity in the absence of inflammation. We hypothesize that virus induced airway hyperreactivity and loss of M muscarinic receptor function are mediated by the production of IFNs. We propose the following specific aims:
SPECIFIC AIM #1 : To determine the effects of exogenous IFNs (alpha, beta, and gamma) on neuronal M2 receptors in vivo.
SPECIFIC AIM #2 : To investigate the role of IFNs in mediating hyperreactivity and loss of neuronal M2 muscarinic receptor function after viral infection and treatment with double-stranded RNA. Animals will be infected with parainfluenza virus or treated with dsRNA, and neutralizing antibodies to various IFNs and their receptors will be used to try to prevent hyperreactivity and M2 receptor dysfunction.
SPECIFIC AIM #3 : To investigate the effects of IFNs on M2 receptor function and gene expression in primary cultures of airway parasympathetic neurons and human neuroblastoma cells. Real time RT-PCR will be used to measure M2 receptor mRNA. Immunofluorescence will be used to measure M2 receptor protein. Stimulated release of acetylcholine and the ability of atropine to potentiate acetylcholine release (by blocking M2 receptors) will be measured to assess M2 receptor function. The neurons' repertoire of interferon regulatory proteins, and the requirement of new protein synthesis will be determined SPECIFIC AIM #4: To use M2 muscarinic receptor promoter reporter constructs to investigate the mechanisms by which IFNs decrease M2 receptor expression. Deletion constructs and site-directed mutagenesis will be used to identify specific promoter elements involved in IFN suppression of M2 receptor gene expression. Particular attention will be paid to IRF-E sites at -43 and -1147 BP, multiple C/EBPbeta sites, and a STAT-1 site at -2754 BP. Etectrophretic mobility shift assays will be used to detect activation of the relevant transcription factors and will allow correlation with functional effects on gene expression.
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