Our laboratory has long been interested in understanding the biology of macrophage activation, the role the macrophage plays in disease processes, and on how understanding the interplay between these two processes may yield information useful to develop newer, better, and safer treatment options. We have focused our investigations on the interaction of monocytes and macrophages with extracellular mediators of inflammation. One research program examines the mechanism of regulation of the cellular response to IFNg - the predominant molecule in T cell supernatants that induce tumoricidal and bacteriocidal activity in macrophages. Immune complexes, for example, have long been known to be capable of altering the cellular response of monocytes or macrophages to IFNg. With the current understanding of the mechanisms by which IFNg induces its signal through the JAK/STAT signalling pathway, the know-how and the methodologies are now available to determine how these signaling pathways could be regulated on the molecular level by components of the extracellular milieu such as immune complexes. A second research area examines the cellular and molecular responses of monocyte-derived dendritic cells when exposed to fragments of the prion protein. Prions are thought to be a potential source of infection for mad cow disease and other types of spongiform encephalopathies - an area of vital regulatory concern given the prevalence of bovine and human sourced materials in the production process of therapeutic biologics. We are studying the ability of prion peptides to bind to either monocytes or dendritic cells, in an attempt to identify the receptor or receptors involved. The subsequent activation of the cell by this receptor binding is also under investigation, in an attempt to gain a better perspective on the mechanisms of prion propagation and inflammation, as well as to explore possibilities of earlier detection of infected or affected tissues. Immune complex-mediated modulation of inflammation. Immune complexes (ICs) modulate antigen driven immune responses in part by their ability to inhibit interferong (IFNg) - inducible activation of inflammatory cells. Many genes, including MHC class II antigens, transcriptionally activated by IFNg require the tyrosine phosphorylation of the transcription factor p91 (Stat1). We have previously shown that this IC-mediated modulation of the IFNg response was due to the inhibition of p91 phosphorylation and the concomitant inhibition of nuclear transcription. Our current studies are extending these initial reports by demonstrating the role of ICs in downregulating the phosphorylation state of the kinases JAK1 and JAK2, as well as the a and b components of the IFNg receptor. The mechanism of this down-regulation appears to involve the recruitment of the phosphatase SHP-1, specifically through the FcgRI signalling pathway. These data elucidate the signalling pathway by which ICs may exert some of their immunomodulatory effects on inflammatory cells, and identify a target for pharmaceutical intervention to prevent these effects. Prion regulation of dendritic cell function. The prion protein (PrP), which is thought to be the infectious agent in the fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), is expressed on many cell types including macrophages. Specific peptides derived from the PrP sequence have been examined for their potential cytotoxic and neurotoxic effects when used in vitro or in-vivo. One such peptide, the PrP106-126 fragment has been shown to mimic the pathologic isoform of prion protein and to induce a cytotoxic effect in neuronal cells. We are characterizing the role of monocytes and monocyte-derived dendritic cells in this process and determining the potential mechanism whereby this cytotoxicity can occur, by determining the ability of PrP106-126 to activate specific signalling pathways in monocytes and dendritic cells derived from elutriated human monocytes. Our data demonstrate that dendritic cells exposed to PrP106-126 undergo a rapid activation of intrinsic NFkB, as demonstrated by electrophoretic mobility shift assays. This activation of NFkB has been correlated with increases in both inflammatory cytokine messenger RNA and protein expression. We have also determined that the signaling pathway through which this activation occurs utilizes p38 and JNK, is probably not G-protein linked, but rather utilizes distinct receptors and a unique signalling pathway that may be Toll-receptor related. These data demonstrate the ability of prion protein fragments to induce inflammatory cytokines through the NFkB signalling pathway and suggest a potential mechanism by which prion proteins may exert their neuro- and cytotoxicity. This project incorporates FY2002 projects 1Z01BO004008-02 and 1Z01BO004009-02.
