The primary objective of the proposed studies is the elucidation of the diverse mechanisms by which the neuropeptide (NP) substance P (SP) modulates immunologic responses through effects on human lymphocytes (HL), and the role that lymphocytes expressing SP receptors (SPR) play in regulating cell mediated immune responses in specific tissues. SP is present at elevated concentrations in tissues at sites of inflammation and is known to modulate connective tissue repair processes and acute hypersensitivity responses.
The specific aims of the proposal encompass an integrated assessment of the proliferative and synthetic responses of the SPR+ HL subset to diverse immunologic stimuli such as antigens, lectins, interleukins (ILs), inflammatory mediators and NP, the chemotactic responses of SPR+ HL, and the role of this subset in cell-cell regulatory interactions. SPR+ HL3 proliferative responses to the above stimuli will be assessed by the uptake of (3H)thymidine, (3H)leucine, and the cell-cycle distribution of these cells as assessed in a FACS. The synthetic responses of this subset will be evaluated by quantifying their immunoglobulin (Ig) production by radioimmunoassay measurements, and their synthesis of IL 2 in an IL 2-dependent cytolytic lymphocyte proliferation assay. SPR+HL chemotactic responses will be assessed by their migration to the above stimuli in micro-Boyden chambers. The current proposal will assess in detail the structural characteristics of the SPR protein. The SPR protein will be further purified by affinity chromatography techniques using an anti-SPR antibody, and cleaved with CNBr, trypsin, and Staph V8 protease to study its subunit structure and to obtain substituent SPR peptides that will be purified over HPLC and sequenced. Antibodies to these peptides will be generated in order to further analyze the SPR structure and to facilitate molecular biology applications. The cDNA for the SPR gene in HL will be identified by constructing oligonucleotide probes based on the above obtained amino acid sequences, probing an IM-9 lymphoblast cDNA library that the investigator has constructed in the vector lambda gtll, expanding the positive clones, and subcloning them into the vector M13 in order to sequence the SPR gene by the dideoxy chain termination procedure. Northern and Southern blots of IM-9 cell RNA and DNA, respectively, will assess the regulation of the HL SPR gene by immunologic stimuli. These integrated functional, structural, and genetic studies of the HL SPR will elucidate the regulation at the molecular level of local tissue immune responses by NP.
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