Bacterial lipopolysaccharide (LPS)-stimulated macrophage tumor Necrosis factor alpha (TNFalpha) production contributes to myocardial depression, shock and multiple organ failure. LPS induces Macrophage TNFalpha production primarily through activation of nuclear factor kappaB (NFkappB) which translocates into the nucleus and activates TNFalpha gene transcription. Thus, NFkappaB represents a target for anti-inflammation therapy. Unfortunately, specific inhibition of NFkappaB is not clinically accessible. Expression of heat shock protein 72 (HSP72) in cardiac Macrophage is associated with enhanced cardiac functional resistance to LPS, suggesting that HSP72 may down-regulate Macrophages response to LPS stimulation. Indeed, treatment of macrophages in vitro by heat attenuates LPS-stimulated TNFalpha production. However, heat stress, like other heat shock response inducers, not only induce HSP72, but also cause other genetic, metabolic, functional and structural alterations. The role of HSP72 itself in the regulation of TNFalpha production remains undefined. Although gene transfer has been used for overexpression of HSP72 in cells and tissues, direct delivery of HSP72 into cells can avoid sustained overexpression of the transferred gene and potential side effects of viral vectors on the host genome. Defining the role and mechanism of HSP72 in the regulation of TNFalpha production may provide insights into the immunological function of this protein and into new therapeutic strategies for the treatment of inflammatory diseases by selective immunosuppression. We hypothesized that HSP72 suppresses LPS-stimulated Macrophage TNFalpha production through contact inhibition of the intranuclear translocation and/or DNA binding activity of NFkappaB. We will characterize heat stress- induced HSP72 expression in cultured macrophage and define the influences of heat shock response on LPS-stimulated NFkappa B activation (translocation and DNA binding) and TNFalpha production (Specific Aim #1). To define the role of HSP72 itself, we will develop a liposomal delivery system to introduce recombinant HSP72 into cultured macrophage (Specific Aim#2). The influences of HSP72 on LPS-stimulated NFkappaB activation and TNFalpha production will be assessed and contrasted to those of the heat shock response (Specific Aim #3). Additional efforts will seek to define the molecular interaction of HSP72 with NFkappaB subunits (p65 and IkappaBalpha) utilizing immuno-colocalization and immunoprecipitation (Specific Aim #4).
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