Abstract
Abstract 3220
Neutrophils play a vital role in innate immunity. Activated neutrophils can release proteolytic enzymes capable of neutralizing microbes and contributing importantly to host-defense. In severe sepsis, microbial components and pro-inflammatory cytokines can contribute to excess systemic neutrophil activation, resulting in tissue damage and organ failure. Thus, regulation of neutrophil activation and factor release is critical during pathologic conditions.
Recent data indicate that components of the contact system modulate numerous inflammatory mediators during severe sepsis, but the exact role of the contact pathway in host-defense is not well understood. Inhibition of factor XII (FXII) in septic baboons reduces circulating neutrophil elastase (NE), a potent cytolytic enzyme that is increased during sepsis and implicated in organ failure. In vitro studies also indicate that both plasma kallikrein and FXIIa are capable of directly inducing NE release. While it is apparent that factors of the contact system interact with neutrophils, the molecular mechanisms by which these factors modulate neutrophil function have not been established. We therefore examined factor XI (FXI) neutrophil interactions and the cellular signaling pathways regulating FXIIa neutrophil stimulation.
Human neutrophils were isolated from peripheral blood and resuspended in HBSS at a concentration of 0.5 ×106/ml. Cells were treated with FXI, FXIa, FXII, or FXIIa with or without fMLP (1 μM) stimulation, and the release of NE was assayed in the cell supernatants via ELISA. FXI, FXIa or FXII had no direct stimulatory effect on NE release compared to vehicle. While neither FXI nor FXII had any inhibitory effect on fMLP induced NE release, FXIa (10 μg/ml) modestly reduced fMLP-induced NE release by 20% (n=3). FXIIa (3, 10, 30 μg/ml) dose-dependently increased NE release in the presence of cytochalasin B (5 μg/ml), consistent with published data.
To examine the mechanism by which FXIIa induces NE release, neutrophils were pretreated with signaling inhibitors and subsequently activated with FXIIa (30 μg/ml). Mammalian target of rapamycyin (mTOR) is a downstream serine/threonine kinase of the PI3K/AKT pathway that integrates signals from the microenvironment such as cytokines and growth factors. It is known that inhibition of mTORC2 abrogates neutrophil polarization and directed migration, thus we examined the role of rapamycin complex 1 and 2 (mTORC1/2) in mediating NE release. Pretreatment of cells with RAD001 (20 nM), an mTORC1 inhibitor had no effect on FXIIa-induced NE release, whereas the combined mTORC1/mTORC2 inhibitor, pp242 (100 nM) abrogated FXIIa-induced NE release, suggesting that components of the mTORC2 pathway contribute to NE release. Pretreatment with EHT 1864 (50 uM), a Rac inhibitor, significantly potentiated NE release induced by either fMLP or FXIIa, suggesting that Rac is also capable of modulating FXIIa signaling.
Taken together, these results suggest that coagulation factors FXIa and FXIIa differentially modulate neutrophil function, and that the mTOR and Rac signaling pathways participate in FXIIa stimulated neutrophil activation. These data suggest that the contact pathway is involved in neutrophil stimulation through mTOR and Rac signaling, and thus modulating these pathways could be a potential therapeutic strategy for limiting excess neutrophil activation.
Disclosures:
Gruber: Aronora, LLC: Consultancy, Equity Ownership.
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