Endotoxin tolerance in sepsis: concentration-dependent augmentation or inhibition of LPS-stimulated macrophage TNF secretion by LPS pretreatment.

Background: We previously showed that macrophages (MΦ) 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 MΦ 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 MΦ responses. Methods: RAW 264.7 MΦ-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 MΦ 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. Results: 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. Conclusion: Prior exposure of MΦ to bacterial ligands alters MΦ cytokine production in response to subsequent LPS-stimulated activation. This modulated MΦ response is critically dependent on the concentration of LPS pretreatment.