The cellular and molecular mechanisms regulating oxidative stress-induced priming of the macrophage
Subtitle: the role of the Src family of tyrosine kinases.
Published: 2004
Language: eng
Number of pages: 201
Description: Civilian trauma remains a leading cause of disability and mortality in North America. Late deaths are related to the development of multiple organ failure in up to two-thirds of these patients. Many studies suggest that the global ischemia-reperfusion resulting from resuscitated hemorrhagic shock predisposes patients to organ injury by priming for an exaggerated immune response to a delayed inflammatory stimulus. The synergistic effect of sequential stimuli has been termed the "two-hit" hypothesis for the development of organ injury in trauma patients.Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. While oxidants are themselves known to induce several intracellular signaling cascades, the affect oxidants have on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work suggested that oxidants can prime alveolar macrophages for increased NF-kappaB translocation in response to treatment with LPS. Since oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-kappaB translocation by LPS via the involvement of Src family kinases.Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets, such as the Src kinase family, for immunomodulation following ischemia/reperfusion.These findings suggest oxidative stress might divert LPS signaling along an alternate signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment activated the of PI3-kinase/Akt pathway. Involvement of this pathway appears to be independent of traditional LPS signaling.To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress can augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-kappaB translocation. Inhibiting Src family kinases either pharmacologically using PP2 or through a molecular approach by cell transfection with carboxyl-terminal Src kinase (Csk) prevents the augmented LPS-induced NF-kappaB translocation caused by oxidants. Interestingly, while inhibiting Src kinases prevented the LPS-induced NF-kappaB translocation in oxidant-treated macrophages, the strategy had no effect on NF-kappaB translocation caused by LPS in the absence of oxidants.Using our in vivo rodent model of hemorrhagic shock/resuscitation we found oxidant stress generated during shock/resuscitation in vivo activates Src family kinases in alveolar macrophages. Inhibiting Src activation by PP2 attenuates alveolar macrophage priming for increased LPS responsiveness following hemorrhagic shock resulting in a modest reduction in lung injury.
Notes: Adviser:  Ori D. Rotstein.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 4992.