NFκB is persistently activated in continuously stimulated human neutrophils

Abstract

Increased activation of the transcription factor NFκB in the neutrophils has been associated with the pathogenesis of sepsis, acute lung injury (ALI), bronchopulmonary dysplasia (BPD), and other neutrophil-mediated inflammatory disorders. Despite recent progress in analyzing early NFκB activation in human neutrophils, activation of NFκB in persistently stimulated neutrophils has not been previously studied, Because it is the persistent NFκB activation that is thought to be involved in the host response to sepsis and the pathogenesis of ALI and BPD, we hypothesized that continuously stimulated human neutrophils may exhibit a late phase of NFκB activity. The goal of this study was to analyze the NFκB activation and expression of IκB and NFκB proteins during neutrophil stimulation with inflammatory signals for prolonged times. We demonstrate that neutrophil stimulation with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNFα) induces, in addition to the early activation at 30-60 min, a previously unrecognized late phase of NFκB activation, In LPS-stimulated neutrophils, this NFκB activity typically had a biphasic character, whereas TNFα-stimulated neutrophils exhibited a continuous NFκB activity peaking around 9 h after stimulation. In contrast to the early NFκB activation that inversely correlates to the nuclear levels of IκBα, however, in continuously stimulated neutrophils, NFκB is persistently activated despite considerable levels of IκBα present in the nucleus. Our data suggest that NFκB is persistently activated in human neutrophils during neutrophil-mediated inflammatory disorders, and this persistent NFκB activity may represent one of the underlying mechanisms for the continuous production of proinflammatory mediators.