Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B

Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g., lipopolysaccharide or LPS) le ads to widespread organ damage and death. The present study demonstrates fo r the first time that the activation of Rac1 (a GTP-binding protein), and t he subsequent production of ROS, constitutes a major pathway involved in NF kappaB-mediated tumor necrosis factor-alpha (TNF alpha) secretion followin g LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1. (N17Rac1) reduced Rac1 activation, ROS formation, NF kappaB activati on, and TNF alpha secretion following LPS stimulation. In contrast, express ion of a dominant active form of Rac1 (V12Rac1) mimicked these effects in t he absence of LPS stimulation. IKK alpha and IKK beta were both required do wnstream modulators of LPS-activated Rac1, since the expression of either o f the IKK dominant mutants (IKK alpha KM or IKK beta KA) drastically reduce d NF kappaB-dependent TNF alpha secretion. Moreover, studies using CD14 blo cking antibodies suggest that Rac1 induces TNF alpha secretion through a pa thway independent of CD14. However, a maximum therapeutic inhibition of LPS -induced TNF alpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependen t pathways could be a useful therapeutic strategy for attenuating the proin flammatory cytokine response during the course of sepsis.