To correlate cytokine gene expression with the release of protein product by murine peritoneal macrophages rendered tolerant by sequential endotoxin stimulation in vitro.
In vitro investigation of the regulation of endotoxin-stimulated cytokine production following endotoxin pretreatment using cytokine bioassays, polymerase chain reaction, and Northern blot analyses.
In vitro cell culture model of sequential endotoxin stimulation of murine macrophages.
Macrophages were pretreated with 0 or 100 ng/mL of lipopolysaccharide (LPS1) for 24 hours and then stimulated with 0 or 100 ng/mL of LPS (LPS2) for 4 or 24 hours. After stimulation, supernatant tumor necrosis factor (TNF) and interleukin-1 (IL-1) levels were measured by bioassay. Total RNA was extracted and messenger RNA (mRNA) corresponding to TNF and IL-1 was amplifed by reverse transcription-polymerase chain reaction or analyzed by Northern blot.
Endotoxin pretreatment resulted in the augmentation of IL-1 (mean±SD, 78±9 vs 596±42 pg/mL, P<.01) and the inhibition of TNF (274±63 vs 61±3 pg/mL, P<.01) release 4 hours after stimulation with 100 ng/mL of LPS2. A similar pattern of cytokine release was observed 24 hours after LPS2 stimulation. Pretreatment produced an increased IL-1 message in response to 100 ng/mL of LPS2. The TNF message was detectable in all groups receiving LPS2 alone, but the highest levels of TNF mRNA were seen in LPS1-pretreated cells stimulated with LPS2.
Endotoxin pretreatment produced increased IL-1 message that paralleled the augmentation of IL-1 protein, whereas abundant TNF message was present even though TNF protein release was significantly inhibited. In this model of in vitro endotoxin tolerance, pretreatment initiates divergent pathways of cytokine regulation.(Arch Surg. 1994;129:1263-1270)
Seatter SC, Bennet T, Li MH, Bubrick MP, West MA. Macrophage Endotoxin ToleranceTumor Necrosis Factor and Interleukin-1 Regulation by Lipopolysaccharide Pretreatment. Arch Surg. 1994;129(12):1263-1270. doi:10.1001/archsurg.1994.01420360053006