Rationale: Indirect acute lung injury (ALI) is associated with high morbidity and mortality. No specific therapies have been developed, because the underlying pathophysiological processes remain elusive. Objectives: To investigate the contribution of Fas-induced apoptotic and nonapoptotic/inflammatory signaling to the pathology of indirect ALI. Methods: A mouse model of indirect ALI, induced by successive exposure to hemorrhagic shock and cecal ligation and puncture, was used. Quantification of active caspase-3 and the short splice variant of FLICE-inhibitory protein, (FLIP) short , was performed by Western blotting and immunohistochemistry, and cytokines/chemokines were assessed by cytometric bead array or ELISA. M30 immunostaining was done to evaluate epithelial cell apoptosis. Lung injury was assessed on the basis of myeloperoxidase activity, bronchoalveolar lavage protein, and lung histology. Measurements and Main Results: Twelve hours after insult, lung monocyte chemoattractant protein-1, keratinocyte-derived chemokine, macrophage inflammatory protein-2, IL-6, tumor necrosis factor-a, and caspase-3 were increased and FLIP short was decreased. Fas-and Fas ligand-deficient mice showed marked protection from lung inflammation and apoptosis and decreased ALI. This was associated with a 10-day survival benefit. Similarly, 4 hours after pulmonary instillation of Fas-activating antibody in vivo, lung chemokines were markedly elevated in background mice and, interestingly, to a similar degree in macrophage-deficient animals. Fas activation on lung epithelial cells in vitro led to chemokine production that was dependent on extracellular signal-regulated kinase. Conclusions: Activation of apoptotic and nonapoptotic/inflammatory Fas signaling is an early important pathophysiological event in the development of indirect ALI after hemorrhagic shock and sepsis, in which lung epithelial cells appear to play a central role.

Objective Indirect acute lung injury (ALI) is associated with high morbidity/ mortality. However, the underlying pathophysiology is only marginally understood and so far no pathophysiologic based remedy exists. We hypothesized, that apoptosis of lung epithelial cells is a pathophysiological relevant process in the development of indirect ALI and that it should be accessible to a small interfering (si)RNA based therapeutic intervention in vivo. Design Prospective, randomized, controlled animal study. Setting Basic science laboratory of a university affiliated level-1 trauma center. Subjects Male C3H/HeN mice, 8 weeks old, n=121 Interventions First, siRNA sequences to knock down caspase-3 expression at a RNA as well as protein level were evaluated in vitro. Then, C3H/HeN mice were subjected to hemorrhagic shock, following which they received either a caspase-3 siRNA or a control/nonsense siRNA. Subsequently, they were then subjected to polymicrobial sepsis (induced by cecal ligation and puncture). Measurements and Main Results Twelve and 24 hours after sepsis increased lung epithelial apoptosis, as evidenced by active caspase-3 western blotting, caspase-3-, TUNEL- and M30 immunohistochemistry, was observed. Hallmarks of ALI, such as increased concentrations of pulmonary cytokines/chemokines, lung protein leakage, myeloperoxidase activity and altered lung histology, were evident in response to these insults. The single intratracheal instillation of caspase-3 siRNA not only attenuated lung apoptosis and inflammation, but also ameliorated the development of ALI in treated mice. Most interestingly, this experimental therapeutic approach markedly improved 10 day survival of hemorrhaged-septic mice. Conclusions Apoptosis of lung epithelial cells is a relevant pathomechanism in the development of hemorrhage-induced indirect septic ALI, and caspase-3 appears to be a valuable therapeutic target, accessible by siRNA treatment in vivo.