Signaling mechanisms of neutrophil apoptosis modulation after burn injury.

摘要

Polymorphonuclear neutrophils (PMNs) play a critical role in defense against invading microbial pathogens in injured and/or infected hosts. On the other hand, an exaggerated neutrophil activation can damage normal tissue. PMN apoptosis is critical to the resolution of acute inflammation and prevention of secondary tissue injury. Previous studies have shown that a delay in PMN apoptosis occurs following burn injury. Such a delay could contribute to failure of resolution of burn inflammation, and promote PMN mediated normal tissue injury. Until now, there has been little information available regarding intracellular events and signaling pathways governing the delay in neutrophil apoptosis that is seen following burn injury. The overall objective of this dissertation was to elucidate cellular mechanisms that contribute to the modulation of neutrophil apoptosis in burn-injured rats. The hypothesis of this project was that burn injury modulates the mitochondrial pathway of neutrophil apoptosis through up-regulation of PI-3 kinase/PKB and NF-kappaB signaling leading to induction of anti-apoptotic Bcl-xl and inhibitory apoptosis protein (IAP) family members. In this dissertation project, the modulation of apoptosis in neutrophils isolated from burn-injured animals was investigated. The potential role of the mitochondrial pathway in such modulations was determined by examining mitochondrial morphological changes that are characteristic of apoptosis. The involvement of pro- and anti-apoptotic Bcl-2 family members was also analyzed. The PI-3K/PKB signaling pathway of PI-3 K/PKB was evaluated to determine if it plays a role in the modulation of PMN apoptosis seen with burn injury. Several downstream events of PI-3 K/PKB activation, including phosphorylation of Bad, NF-kappaB activation, and gene/protein expression of IAP family members were also examined to ascertain if they are related to the modulation of mitochondrial mechanisms of neutrophil apoptosis with burn injury. The results showed that neutrophil apoptosis is delayed in rats subjected to burn injury. In neutrophils from sham rats, mitochondria showed a change from normal "tubular" to an "aggregated" morphology during incubation. In contrast, neutrophils from burn rats did not exhibit this mitochondrial transition until 8-hr of incubation. Whereas an up-regulation of Bcl-xl and a down-regulation of Bax seemed to contribute to decreased apoptosis in neutrophils from burn rats at 2 hr of incubation, the decreased apoptosis at 8 hr appeared to be associated with a decrease in Bcl-xl expression along with up-regulation of phosphorylated Bad. These data suggest that suppression of the mitochondrial pathway plays an essential role in the delay of PMN apoptosis that occurs with burn injury. The delay of neutrophil apoptosis with burn injury is likely caused by the activation of PI-3 kinase/PKB signaling pathway, which is upstream of phosphorylation of Bad and NF-kappaB activation. The anti-apoptosic effect of NF-kappaB with burn injury depends upon increased expression of Bcl-xl, but not IAPs. Moreover, increased Bcl-xl expression and Bad phosphorylation, which are dependent on PI-3 kinase activation, appear to operate independently to affect the delay of PMN apoptosis following burn injury.