ABSTRACT
Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these important changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.
ABSTRACT
Activation of Fas signaling is a potentially important pathophysiological mechanism in the development of septic acute lung injury (ALI). However, so far the optimal targets within this signaling cascade remain elusive. Thus, we tested the hypothesis that in vivo gene silencing of Fas, Fas-associated via death domain (FADD), or caspase 3 by intratracheal administration of small interfering RNA would ameliorate ALI in a clinically relevant double-hit mouse model of trauma induced septic lung injury. Male C57Bl/6 mice received small interfering (Fas, FADD, caspase 3) or control RNA 24 h before and 12 h after blunt chest trauma or sham procedures. Polymicrobial sepsis was induced by cecal ligation and puncture 24 h after chest trauma. Twelve or 24 h later, lung tissue, plasma, and bronchoalveolar lavage fluid were harvested. During ALI, lung apoptosis (active caspase 3 Western blotting, TUNEL staining) was substantially increased when compared with sham. Silencing of caspase 3 or FADD both markedly reduced pulmonary apoptosis. Fas- and FADD-small interfering RNA administration substantially decreased lung cytokine concentration, whereas caspase 3 silencing did not reduce lung inflammation. In addition, Fas silencing markedly decreased lung neutrophil infiltration. Interestingly, only in response to caspase 3 silencing, ALI-induced lung epithelial barrier dysfunction was substantially improved, and histological appearance was beneficially affected. Taken together, downstream inhibition of lung apoptosis via caspase 3 silencing proved to be superior in mitigating ALI when compared with upstream inhibition of apoptosis via Fas or FADD silencing, even in the presence of additional anti-inflammatory effects. This indicates a major pathophysiological role of lung apoptosis and suggests the importance of other than Fas-driven apoptotic pathways in trauma-induced septic ALI.
