Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-κB, and expression of the proinflammatory cytokines IL-1β and TNF-α through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110γ catalytic subunit of PI3-K. In PI3-Kγ−/− mice, lung edema, neutrophil recruitment, nuclear translocation of NF-κB, and pulmonary levels of IL-1β and TNF-α were significantly lower after endotoxemia as compared with PI3-Kγ+/+ controls. Among neutrophils that did accumulate in the lungs of the PI3-Kγ−/− mice after endotoxin administration, activation of NF-κB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kγ+/+ mice. These results show that PI3-K, and particularly PI3-Kγ, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.

The transcriptional regulatory factor nuclear factor (NF)-κB has a central role in modulating expression of proinflammatory mediators that are important in acute lung injury. In vitro studies have shown that competition between NF-κB and cAMP response element binding protein (CREB) for binding to the coactivator CREB-binding protein (CBP) is important in regulating transcriptional activity of these factors. In the present study, we examined in vivo interactions between CBP, CREB, and NF-κB in hemorrhage- or endotoxemia-induced acute lung injury. Association of CBP with CREB or the p65 subunit of NF-κB increased in the lungs after hemorrhage or endotoxemia. Inhibition of xanthine oxidase before hemorrhage, but not before endotoxemia, decreased p65-CBP interactions while increasing those between CREB and CBP. These alterations in CREB-CBP and p65-CBP interactions were functionally significant because xanthine oxidase inhibition before hemorrhage resulted in increased expression of the CREB-dependent gene c-Fos and decreased expression of macrophage inflammatory protein-2, a NF-κB-dependent gene. The present results show that the coactivator CBP has an important role in modulating transcription in vivo under clinically relevant pathophysiological conditions.

Acute lung injury is frequently associated with endotoxemia and is characterized by the accumulation in the lungs of large numbers of neutrophils activated to produce proinflammatory mediators. In the setting of acute lung injury, the percentage of apoptotic cells among lung neutrophils is decreased. The transcriptional regulatory factor NF-κB is activated in neutrophils and other pulmonary cell populations after endotoxemia and appears to play a central role in the development of the acute inflammatory process that leads to lung injury. Because NF-κB can modulate apoptosis through increasing expression of anti-apoptotic proteins, activation of NF-κB may contribute to the alterations in lung neutrophil apoptosis associated with acute lung injury. In the present experiments, endotoxemia resulted in decreased apoptosis and increased expression of anti-apoptotic mediators among lung neutrophils. Amounts of A1, A20, and Bcl-xL, anti-apoptotic proteins whose transcription is dependent on NF-κB, were increased in lung neutrophils after endotoxemia. Inhibition of nuclear translocation of NF-κB increased the percentage of apoptotic lung neutrophils after endotoxemia, but not back to the levels found in unmanipulated animals. Although inhibition of nuclear translocation of NF-κB prevented endotoxemia-induced increases in Bcl-xL, A1, and A20 in lung neutrophils, this intervention did not prevent endotoxemia-associated elevation of Mcl-1, an anti-apoptotic protein primarily under the transcriptional regulation of CREB. These results demonstrate that mechanisms independent of NF-κB activation play an important role in modulating lung neutrophil apoptosis after endotoxemia,

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