Endotoxin Tolerance in Sepsis: Concentration-Dependent Augmentation or Inhibition of LPS-Stimulated Macrophage TNF Secretion by LPS Pretreatment

Abstract

We previously showed that macrophages (MPhi) pretreated with bacterial endotoxin (lipopolysaccharide [LPS]) develop an altered state of LPS-responsiveness--"LPS tolerance": LPS tolerance was associated with inhibition of tumor necrosis factor (TNF) release and decreased extracellular signal-regulated kinase and p38 kinase activation when MPhi were restimulated with LPS. However, the concentration of LPS used for pretreatment (most frequently 10 ng/mL) may be much higher than LPS concentrations observed in patients. Therefore, in the current study we examined the effect of lower and higher pretreatment LPS concentrations on subsequent LPS-stimulated MPhi responses. RAW 264.7 MPhi-like cells were pretreated in vitro (PreRx) for 24 hours in medium or a range of LPS concentrations (0 ng/mL, 1 ng/mL, 10 ng/mL, or 100 ng/mL of E. coli 0111B4 LPS). Culture medium was discarded after 24 hours and MPhi were restimulated with LPS (0 ng/mL, 1 ng/mL, 10 ng/mL or 100 ng/mL). Three different lots of LPS (Sigma) were used. Supernatant TNF secretion at 3 hour was measured using enzyme-linked immunosorbent assay (pg/mL +/- SEM). Statistics by Chi-square and student's t test. Pretreatment with 100 ng/mL of LPS profoundly inhibited TNF release at all LPS restimulation concentrations (p < 0.05 vs. Medium PreRx). In contrast, very low dose LPS pretreatment (1 ng/mL) significantly augmented TNF release versus medium (p < 0.05). There was no further augmentation observed when even lower doses of LPS (0.1 ng/mL) were used for pretreatment. Similar results were obtained with three different lots E. coli 0111B4 LPS or using LPS from E. coli 0127B8. Prior exposure of MPhi to bacterial ligands alters MPhi cytokine production in response to subsequent LPS-stimulated activation. This modulated MPhi response is critically dependent on the concentration of LPS pretreatment.