- Meeting abstract
Modulation of NF-κB signaling in macrophages under long-term and second-hit stimulation
Critical Care volume 7, Article number: P044 (2003)
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 , while being inalienable for the inflammation process . 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.
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.
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.
The LPS tolerance  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.
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Groesdonk, H., Baur, A., Reithmeier, E. et al. Modulation of NF-κB signaling in macrophages under long-term and second-hit stimulation. Crit Care 7, P044 (2003). https://doi.org/10.1186/cc1933
- Electrophoretic Mobility Shift Assay
- Multiple Organ Dysfunction Syndrome
- Nuclear Protein Extract
- Impaired Control
- Critical Disease