Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human monocytes.
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.


CONCLUSIONS
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.
Heagy
W
W
Department of Surgery, Hennepin County Medical Center, Minneapolis Medical Research Foundation, University of Minnesota 55415, USA.
Hansen
C
C
Nieman
K
K
Rodriguez
J L
JL
West
M A
MA
D016207:Cytokines / Q000032:analysis* / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation; D008070:Lipopolysaccharides / Q000276:immunology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000276:immunology*; D009000:Monocytes / Q000378:metabolism*; D009102:Multiple Organ Failure / Q000209:etiology; D009104:Multiple Trauma / Q000150:complications / Q000276:immunology; D015398:Signal Transduction; D018746:Systemic Inflammatory Response Syndrome / Q000209:etiology
D016207:Cytokines; D008070:Lipopolysaccharides; D020929:Mitogen-Activated Protein Kinase Kinases