Multiorgan failure is a major cause of late mortality following
trauma. Oxidative stress generated during
shock/
resuscitation contributes to tissue injury by priming the immune system for an exaggerated response to subsequent inflammatory stimuli, such as
lipopolysaccharide (LPS). We recently reported that oxidative stress causes rapid recruitment of the
LPS receptor Toll-like receptor 4 (TLR4) to
membrane lipid rafts, thus increasing LPS responsiveness and cellular priming. We hypothesized that activation of
Src family kinases by
oxidants might contribute to these events. We utilized microscopy, flow cytometry, Western blotting, and thin-layer chromatography methods. Using
hydrogen peroxide in vitro and
hemorrhagic shock/
resuscitation in vivo,
oxidant-induced TLR4 translocation in macrophages occurred in an Src-dependent manner. Approaches supporting this conclusion included pharmacologic inhibition of the
Src family kinases by PP2, Src inhibition by a molecular approach of cell transfection with Csk, and genetic inhibition of all
Src kinases relevant to the monocyte/macrophage lineage in hckfgrlyn triple knockout mice. To evaluate the upstream molecules involved in Src activation, we evaluated the ability of oxidative stress to activate the bioactive
lipid molecule
ceramide.
Oxidants induced
ceramide generation in macrophages both in vitro and in vivo, an effect that appears to be due to activation of the
acid sphingomyelinase. Using pharmacological approaches,
ceramide was shown to be both necessary and sufficient to mediate TLR4 translocation to the plasma membrane in an Src-dependent manner. This study identifies a hierarchy of signaling molecules following oxidative stress that might represent novel targets for
therapy in
critical illness and organ injury.