Modulation of NF-κB signaling in macrophages under long-term and second-hit stimulation

Introduction

Sepsis often leads to deterioration of organ function, so-called multiple organ dysfunction syndrome (MODS). Pathophysiologically, impaired control mechanisms of NF-κB activation result in a dysbalance between proinflammatory and anti-inflammatory gene transcription. In regard to hyperinflammation, NF-κB activation is thought to play a central role [1], while being inalienable for the inflammation process [2]. Therefore it is conceivable that both proinflammatory and anti-inflammatory cell functions are mediated by NF-κB in the same cell system. The aim of this study is to elucidate the activation kinetics of NF-κB under long-term stimulation (LTS) and two-time stimulation (TTS), simulating conditions in critical diseases.

Methods

Murine macrophages (cell line RAW 264.7) were cultured in standard medium (DMEM + 10% FCS) and incubated in 0.1 μg/ml, 1.0 μg/ml and 10 μg/ml lipopolysaccharide (LPS). After 5, 10, 20, 30, 60 min, and again after 2, 4, 8, 12, 16, and 24 hours, we analyzed the activity of NF-κB in cellular and nuclear protein extracts using the methods of Electrophoretic Mobility Shift Assay (EMSA) and western blots (p65, IκBα). After 4 hours pre-incubation with 0.1 μg/ml, 1.0 μg/ml and 10 μg/ml LPS, we stimulated again a second time with 1 μg/ml LPS (TTS). The analysis mentioned above was done after 5, 10, 20, 30, 60, and 120 min.

Results

Cells incubated with high concentrations of LPS (10 μg/ml) showed a biphasic activation pattern of NF-κB after 5–10 min and 12–16 hours. Cells incubated with lower LPS concentrations showed no further activation after the early activation peak. After preincubation with 0.1 μg/ml LPS and TTS an unaltered timely activation of NF-κB with delayed IκBα degradation was observed.

Conclusion

The LPS tolerance [3] often described in the literature does not exist in macrophages preincubated with low concentrations of LPS. The biphasic activation pattern of the NF-κB signaling pathway induced by high LPS concentrations occurs possibly within the scope of an anti-inflammatory regulation mechanisms. These results clarify the varying activation kinetic of the NF-κB signaling pathway.