Macrophage activation is a two step stimulus-driven process of differentiation that, upon completion, endows the macrophage with the capacity to recognize and destroy neoplastic target cells. As such, macrophage activation represents an important limb of the host defence system. Monocyte derived macrophages are initially primed by exposure to IFN alpha, beta, or gamma. Priming is an essential competence step that allows macrophages to respond to secondary stimuli (e.g. double stranded RNA, zymosan particles, LPS) which trigger the expression of the activated phenotype. The purpose of this proposal is to investigate the mechanisms that positively and negatively regulate the progression of the primed to the activated phenotype. It is hypothesized that a single event, the stimulus induced hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), leads to the production of two second messengers with dichotomous effects on the expression of the activated phenotype. Inositol (1,4,5) triphosphate, by virtue of its ability to initially increase the intracellular concentration of Ca2+ is viewed as positively regulating the expression of the activated state in primed macrophages, while diacylglycerol, which activates protein kinase C, is hypothesized to negatively regulate the expression of the activated state, and may induce further differentiation to the post activated (non tumoricidal) state. IFN priming is viewed as the essential competence step that allows these second messengers to productively regulate the expression of the activated phenotype. These goals will be addressed with three specific aims. The objective of specific aim one is to compare the effects of stimuli of the activated state (poly (I:C) and zymosan particles) on the hydrolysis of PIP2 in unprimed and IFN primed macrophages. Attention will be paid to characterizing any differences in the magnitude or time course of the response that may point to the level of the priming effect. Other experiments will attempt to link the products of PIP2 hydrolysis to the expression of the activated state.
Specific aim two will address the question of roles of extracellular and intracellular Ca2+ in the induction of the activated phenotype, while specific aim three is aimed at determining the role of the enzyme protein kinase C in the negative regulation of the activated phenotype. It is expected that the experiments described herein will allow the level of the priming effect to be deduced. This is predicted to be at a site downstream from an initial rise in intracellular Ca2+. In relation to longer term goals, these studies will set the stage for specific investigations aimed at understanding the molecular mechanism of priming.
