DESCRIPTION (provided by applicant) The acute respiratory distress syndrome (ARDS) is an important clinical problem in the United States, affecting 200,000 patients per year and resulting in death of approximately 75,000 persons. Our laboratory is interested in the role of the Fas/FasL system in the pathophysiology of acute lung injury (ALI) in humans and animal models. This system is comprised of the membrane surface receptor Fas (CD95) and its cognate ligand, FasL (CD178). Binding of Fas to FasL activates signaling pathways that lead to apoptosis and also to cytokine release. We have shown that activation of the Fas/FasL system in animals leads to ALI, whereas mice lacking functional Fas are protected in LPS-induced lung injury. We have also shown that the Fas/FasL system is active in the lungs of humans with ARDS. Recently we have found that the biological activity of sFasL in the lungs is dependent on its structure. Specifically, we determine that sFasL exists in at least two forms: a 144- amino acid short form, consisting primarily of the binding domain, and a 178 amino-acid long form, consisting of the binding domain plus a short juxtamembrane "stalk" region. Only the long form of sFasL, which is the major form that is present in BAL fluid from patients with ARDS. Importantly, we have found that a sFasL mutant in which all 8 charged amino acids of the stalk region have been changed to alanines has decreased function and furthermore, acts as an inhibitor of native sFasL in vitro. We now seek to determine the mechanism of inhibition of the 8-site mutated sFasL and whether it attenuates lung injury in vivo. Hypothesis: The 8-site mutated sFasL acts as an inhibitor of native sFasL by impairing the ability of the Fas receptor to form membrane clusters that are needed to initiate signaling. Therefore, the 8-site mutated sFasL will attenuate the pro-apoptotic activity of human ARDS BALF in vitro and the severity of experimental acute lung injury in vivo. Aim 1: Determine whether the 8-site mutated sFasL inhibits native sFasL by impairing the formation of Fas:FasL clusters and subsequent receptor:ligand capping. Aim 2: Determine whether the 8-site mutated sFasL attenuates the pro-apoptotic activity of BALF from humans with ARDS in vitro. Aim 3: Determine whether the 8-site mutated sFasL attenuates the severity of experimental acute lung injury in vivo. Findings derived from this study will have a transformative impact in the field by refocusing attention on the mechanistic role of pro-apoptotic systems in acute lung injury, and by discovering new potential therapeutic agents for ARDS.