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Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human
1Department of Surgery, Hennepin County Medical Center, Minneapolis Medical Research Foundation, University of Minnesota 55415, USA.
Background:
Dysregulation of monocyte/macrophage cytokine production after exposure to multiple inflammatory stimuli may contribute to multiple organ failure and sepsis. Endotoxin (lipopolysaccharide [LPS]) activation of murine macrophage results in the phosphorylation of kinases in the mitogen-activated protein kinase cascade. Pretreatment of murine macrophages with LPS induces LPS-tolerance, with inhibition of LPS-stimulated activation of kinases (ERK1/2 and p38) and diminished release of tumor necrosis factor (TNF). We sought to determine whether similar alterations in LPS-dependent signal transduction are present in LPS-tolerant human peripheral blood monocytes.
Methods:
Human peripheral blood monocytes from healthy volunteer donors (n = 12) were incubated in RPMI 1640 culture medium +/- 10 ng/mL of LPS for 18 hours, then stimulated with 0 to 1,000 ng/mL of LPS. Supernatant TNF and interleukin-1 (IL-1) levels were measured after 5 hours by enzyme-linked immunosorbent assay. Activation of the p42/p44 kinases (ERK1/2) was measured 15 minutes after LPS with monoclonal antibodies to diphosphorylated (active) ERK1/2 using novel flow cytometric methods.
Results:
LPS-tolerant (10 ng/mL LPS pretreatment) human monocytes had significant inhibition of LPS-stimulated TNF secretion but augmented IL-1 release (p < 0.05). Nontolerant human monocytes had a dramatic increase in the percentage of ERK1/2-positive cells in response to an initial stimulation with LPS. This did not occur in the LPS-tolerant cells. Phorbol-12-myristate-13 acetate restored ERK1/2 activation in LPS-tolerant human monocytes.
Conclusion:
LPS-tolerance in human monocytes is associated with inhibition of LPS-stimulated TNF secretion, augmented release of IL-1, and defective activation of mitogen-activated protein kinase cascade (ERK1/2). These results suggest a method of identifying LPS-tolerance and monocyte dysfunction in patients with sepsis.
Insights
Lipopolysaccharide (LPS)-tolerant human monocytes show reduced tumor necrosis factor (TNF) but increased interleukin-1 (IL-1) release. This tolerance involves defective ERK1/2 activation, suggesting a way to identify monocyte dysfunction in sepsis.
Area of Science:
- Immunology
- Cellular Biology
- Molecular Medicine
Background:
- Monocyte/macrophage cytokine dysregulation contributes to sepsis and organ failure.
- Lipopolysaccharide (LPS) exposure induces tolerance in murine macrophages, inhibiting specific kinase pathways and tumor necrosis factor (TNF) release.
- Investigating LPS-tolerance in human monocytes is crucial for understanding sepsis pathogenesis.
Purpose of the Study:
- To determine if human monocytes exhibit similar LPS-tolerance signaling alterations as observed in murine models.
- To investigate the impact of LPS-tolerance on cytokine production (TNF and IL-1) and mitogen-activated protein kinase (MAPK) pathway activation in human monocytes.
Main Methods:
- Human peripheral blood monocytes were pretreated with LPS to induce tolerance.
- Monocytes were subsequently stimulated with varying LPS concentrations.
- Tumor necrosis factor (TNF) and interleukin-1 (IL-1) levels were measured via ELISA.
- Activation of ERK1/2 kinases was assessed using flow cytometry.
Main Results:
- LPS-tolerant human monocytes showed inhibited TNF secretion but augmented IL-1 release.
- LPS stimulation induced ERK1/2 activation in non-tolerant monocytes, but this was defective in tolerant cells.
- Phorbol ester treatment restored ERK1/2 activation in LPS-tolerant monocytes, indicating pathway reversibility.
Conclusions:
- LPS-tolerance in human monocytes is characterized by suppressed TNF, enhanced IL-1 release, and impaired ERK1/2 activation.
- These findings suggest a potential method for identifying LPS-tolerance and monocyte dysfunction in sepsis patients.
- Understanding these mechanisms could lead to novel therapeutic strategies for sepsis.