Drotrecogin alfa (activated) inhibits NF-kappa B activation and MIP-1-alpha release from isolated mononuclear cells of patients with severe sepsis

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Abstract
Objective and design: Non-anticoagulant biological activities, such as anti-inflammatory and anti-apoptotic mechanisms of action, have been suggested for recombinant human activated protein C (rhAPC; drotrecogin alfa (activated)). However, these mechanisms are much less characterized and understood than rhAPC’s anticoagulant activity. Aim of the study was to determine the effect of rhAPC on the activity of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) in mononuclear cells isolated from septic patients and to characterize an effect downstream from NF-κB activation, such as the release of the NF-κB-controlled chemokine Macrophage Inflammatory Protein-1-alpha (MIP-1-α). Subjects: Peripheral blood was obtained from 13 septic patients and from 8 healthy controls. Methods: Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation and were incubated with or without rhAPC (10 μg/ml) for 2 h for the measurement of NF-κB activity in cell lysates or alternatively for 6 h for the determination of MIP-1-α levels in supernatants. NF-κB activity was measured by an ELISA-based assay directed against the p50 and the p65 subunit of NF-κB. Results: RhAPC, at supra-pharmacological concentration (10 μg/ml), significantly inhibited NF-κB activity and the release of MIP-1-α ex vivo in isolated mononuclear cells from patients with severe sepsis. In mononuclear cells of healthy subjects, however, rhAPC did not change NF-κB activity. Basal NF-κB activity early in severe sepsis was not predictive for survival. Conclusions: RhAPC at supra-pharmacological concentration (10 μg/ml) inhibits the activity of NF-κB in ex vivo isolated mononuclear cells of septic patients as well as the release of MIP-1-α, a proinflammatory chemokine regulated by NF-κB. These findings may represent immunomodulatory pathways by which rhAPC exerts specific anti-inflammatory activity in vitro in addition to its known anticoagulant and profibrinolytic activity and should be further investigated in an in vivo setting.

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