Neuroimmunomodulation of the mucosal immune system is though to exist, but has not been demonstrated. Evidence to support this hypothesis is based on animal studies of systemic and non-mucosal immunocytes. Functional immunomodulation by neuropeptides via specific membrane receptors has been demonstrated in peripheral blood lymphocytes (PBL) and other tissues in animals; no studies have characterized the immune response of mucosal, lamina propria lymphocytes (LPL) to neuropeptide stimulation, in animals or humans. Phenotypic and functional differences in circulating and tissue-based lymphocytes are apparent as well as are differences in levels of neuropeptide concentration. Given these, demonstration of functional neuropeptide receptors on LPL with characteristics different from those in peripheral blood is probable, and would suggest a mechanism for selective modulation of immunity by sensory nerves in human gut mucosa. During Phase 1, PBL from normal healthy donors and LPL from resected intestinal specimens will be examined to determine which subsets express surface receptors for the neuropeptides Substance P (SP) and/or Vasoactive Intestinal Peptide (VIP). Lymphocyte subsets with significant numbers of receptors will be rapidly identified by simultaneous fluorescent activated cell (FACS) analysis using phycoerythrin-conjugated monoclonal antibodies against lymphocyte surface antigens and fluorescein-tagged neuropeptides binding to putative receptors. Focused attention will then be directed toward the identified subsets which will be purified using antibody-panning and complement lysis techniques in order to perform classical radioligand binding studies. Receptor characteristics will be determined. Modulatory effects of neuropeptide stimulation will be measured using appropriate function assays for the identified subset (cytotoxicity, proliferation and immunoglobulin production). Correlations between a subset's receptor characteristics and altered biological activity will be made. The hypothesis of differences in mucosal and systemic neuroimmunomodulation will be determined. During Phase 2, the previous investigations which examined cellular characteristics of neuropeptide-lymphocyte interaction will progress to investigations of the mechanism of sub-cellular activation. In studying a few subsets with marked alteration in function following neuropeptide stimulation, the role of secondary messengers in mediating the receptor-initiated signal will be examined.
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