In Crohn's disease, granulomatous inflammation induces fibrosis. Probably defective immunoregulatory circuits permit the inflammation to persist, causing this tissue damage. Our goal is to treat ineffective or hyper responsive immune responses, such as IBD, by modulating endogenous immunoregulatory pathways through pharmacological manipulation. Neurokines such as substance P (SP), somatostatin 1-14 (SOM14) and vasoactive intestinal peptide (VIP) can modulate a variety of immune functions. Our hypothesis is that SP, SOM released at sites of chronic inflammation regulate important aspects of the immune response. Also proposed is that inflammatory mediators govern neurokine and neurokine receptor expression. We submit that pharmacological manipulation of neurokine immunoregulatory circuits will help control aberrant inflammation. Using murine schistosomiasis mansoni, a chronic granulomatous disease of the liver and intestines, we uncovered a neurokine immunoregulatory circuit within schistosome granulomas. We will investigate further this important discovery with the following three inter-related specific aims: 1. The first specific aim derives from studies suggesting that SP and SOM receptors are inducible on inflammatory cells, which could be an important mechanism regulating neurokine action. We will learn the full extent of SP and SOM receptor distribution among the various granuloma cell subtypes. Also, we will resolve if antigenic stimulation and cytokines induce/modulate neurokine receptor expression. These studies will use competitive binding assays, mRNA analysis and in situ hybridization. The cells of most interest will be cloned for further investigation. 2. The second specific aim develops from our observation that SP receptor blockade selectively and completely inhibits granuloma IgM secretion without affecting granuloma IgG1 production. We want to extend this important observation by learning at what step in B cell development SP regulates granuloma IgM secretion. Also we will find out if SP acts directly on granuloma B cells or indirectly through regulation of cytokines essential for granuloma IgM B cell development. Moreover, we will establish if SP or SOM14 regulates expression of other granuloma antibody isotopes. Methods employed will include ELISPOT assays to count antibody-producing B cells, Ig-specific ELISAs to measure rates of Ig secretion, flow cytometry to count numbers of unswitched verses switched B cells and mRNA analysis of heavy chain useage. 3. The third specific aim stems from the discovery that SP and SOM14 control production of some granuloma lymphokines. We need to learn which cytokines are subject to neurokine control and the mechanisms governing this regulation. Techniques used will include cell culture and cloning, ELISAs and mRNA analysis.
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