During steady-state conditions, heat shock proteins (HSPs) function in the intracellular compartment as molecular chaperones, assisting with the assembly, stabilization, folding, and transport of proteins. Under conditions of stress, HSPs bind and stabilize misfolded proteins and protect them from denaturation, thereby conferring resistance to further stress, an effect known as "stress tolerance." Recent evidence has focused attention on the role that extracellular HSPs play in the host innate immune response as opposed to their more commonly ascribed intracellular chaperone functions. For example, extracellular Hsp70, a member of the 70 kDa family of HSPs plays an important immuno-modulatory role, serving as both a chaperone and cytokine ("chaperokine"). This new paradigm may be explained by the so-called "danger hypothesis", in which stressed cells release Hsp70 into their surrounding extracellular milieu to signal an impending "danger" to neighboring cells. We have shown that stressed monocytes andmacrophages release functional Hsp70 in vitro and that extracellular Hsp70 correlates with poor outcome in children with septic shock. Several questions, however, remain unanswered. Our goals are to further elucidate the mechanisms leading to the extracellular release of Hsp70 and to understand the functional role of extracellular Hsp70 in vitro and in vivo. Accordingly, the central hypothesis of the current proposal is that extracellular Hsp70 modulates important proinflammatory signal transduction pathways in vitro and in vivo. Using a variety of techniques and models, we will determine the conditions and mechanisms leading to release of Hsp70 in vitro and in vivo (Specific Aim 1), determine the effect of extracellular Hsp70 on the innate immune response to components of both gram negative and gram positive bacteria in vitro and in vivo (Specific Aims 2, 3), and determine the effects of extracellular Hsp70 on the innate immune response to polymicrobial sepsis in vivo (Specific Aim 4). The novel finding that an endogenous molecule such as Hsp70 could modulate the host response to a variety of stimuli acting via multiple receptor pathways would provide important insight regarding the functional role of extracellular HSP70 and could ultimately provide a mechanistic link between tissue injury and modulation of the host innate immune response in clinical conditions such as trauma, sepsis, and MODS.