Macrophages are critically important for phagocytosis and killing of microbes, for secreting cytokines to activate leukocytes, and for presenting antigen to MHC compatible T lymphocytes. Since activated macrophages perform these functions with much greater efficiency, understanding the events required to activate these cells is of considerable importance. While cytokines, such as interferon-gamma, have important macrophage activating factors, little attention has been given to the peptide, substance P. This peptide is found in tissues undergoing inflammatory processes and in normal lymphoid tissues. It is also clear that macrophages express substance P receptors. The overall goal of this project is to determine the significance of substance P production and substance P receptor expression by macrophages using in vitro and in vivo models of macrophage activation. Specifically, nuclease protection assays and radioimmunoassays will be performed to quantify preprotachykinin mRNA expression and substance P secretion, respectively. These studies are of extreme importance since they challenge the current view that substance P is solely a product of neuronal cells. The ability of macrophages to secrete substance P and express receptors for this peptide suggest that autocrine mechanisms can occur. Unfortunately, it is not possible to definitively address the role of substance P receptor expression in macrophage activation since reagents to detect this receptor are not available. To this end, the murine macrophage substance P receptor will be sequenced using the dideoxy chain termination method, and cloned receptor fragments used to develop sensitive quantitative competitive-reverse transcribed PCR (QC-RT-PCR). These assays will quantify receptor mRNA expressed by macrophages activated in vitro or in vivo. Using a protein expression system, it will be possible to express recombinant receptor protein for use as an immunogen. Antibodies specific for the macrophage substance P receptor and I-125 labelled substance P will be used to quantify receptor expression on macrophages activated in vitro and in vivo. The ability of activated macrophages to up regulate substance P receptors, strongly suggests a role in macrophage activation. To prove this hypothesis, the effect substance P has on several critical macrophage functions will be determined. These functions include: 1) phagocytosis and killing of Salmonella typhimurium; 2) expression of monokine, class II MHC, and substance P receptor mRNAs as quantified by QC-RT-PCR; 3) secretion of monokines quantified by ELISA and bioassays; and 4) cell surface expression of class II MHC and substance P receptor proteins using immunofluorescence. To prove specificity and significance of the substance P induced effects, antagonists of the response will be used. These antagonists will include: 1) a peptide antagonist (Spantide II); 2) anti-substance P receptor antibodies; and 3) anti-sense oligonucleotides. Ultimately the ability of substance P antagonists to increase the pathogenicity of the intracellular pathogen, Salmonella typhimurium, will be determined using a murine model. Together these studies are likely to demonstrate that the substance P- substance P receptor interaction is one of considerable importance for the activation of macrophages.
Showing the most recent 10 out of 36 publications
