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.

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The immunomodulatory effects of mesenchymal stem cells (MSCs) are established. However, the effects of MSCs on neutrophil survival in acute lung injury (ALI) remain unclear. The goal of this study was to investigate the effect of an MSC-conditioned medium (MSC-CM) on neutrophil apoptosis in endotoxin-induced ALI. In this study, an MSC-CM was delivered via tail vein injection to wild-type male C57BL/6 mice 4 h after an intratracheal injection of lipopolysaccharide (LPS). Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue were collected to perform histology, immunohistochemistry, apoptosis assay of neutrophil, enzyme-linked immunosorbent assays, and an electrophoretic mobility shift assay. Human neutrophils were also collected from patients with sepsis-induced acute respiratory distress syndrome (ARDS). Human neutrophils were treated in vitro with LPS, with or without subsequent MSC-CM co-treatment, and were then analyzed. Administration of the MSC-CM resulted in a significant attenuation of histopathological changes, the levels of interleukin-6 and macrophage inflammatory protein 2, and neutrophil accumulation in mouse lung tissues of LPS-induced ALI. Additionally, MSC-CM therapy enhanced the apoptosis of BALF neutrophils and reduced the expression of the anti-apoptotic molecules, Bcl-xL and Mcl-1, both in vivo and in vitro experiments. Furthermore, phosphorylated and total levels of nuclear factor (NF)-κB p65 were reduced in lung tissues from LPS + MSC-CM mice. Human MSC-CM also reduced the activity levels of NF-κB and matrix metalloproteinase-9 in the human neutrophils from ARDS patients. Thus, the results of this study suggest that the MSC-CM attenuated LPS-induced ALI by inducing neutrophil apoptosis, associated with inhibition of the NF-κB pathway.

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,