MACROPHAGE ENDOTOXIN TOLERANCE TUMOR-NECROSIS-FACTOR AND INTERLEUKIN-1 REGULATION BY LIPOPOLYSACCHARIDE PRETREATMENT

Objective: To correlate cytokine gene expression with the release of p rotein product by murine peritoneal macrophages rendered tolerant by s equential endotoxin stimulation in vitro. Design: In vitro investigati on of the regulation of endotoxin-stimulated cytokine production follo wing endotoxin pretreatment using cytokine bioassays, polymerase chain reaction, and Northern blot analyses. Setting: In vitro cell culture model of sequential endotoxin stimulation of murine macrophages. Inter ventions: Macrophages were pretreated with O or 100 ng/mL of lipopolys accharide (LPS(1)) for 24 hours and then stimulated with 0 or 100 ng/m L of LPS (LPS(2)) for 4 or 24 hours. After stimulation, supernatant tu mor necrosis factor (TNF) and interleukin-1 (IL-1) levels were measure d by bioassay. Total RNA was extracted and messenger RNA (mRNA) corres ponding to TNF and IL-1 was amplifed by reverse transcription-polymera se chain reaction or analyzed by Northern blot. Results: Endotoxin pre treatment resulted in the augmentation of IL-1 (mean+/-SD, 78+/-9 vs 5 96+/-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 LPS(2) . A similar pattern of cytokine release was observed 24 hours after LP S(2) stimulation. Pretreatment prbduced an increased IL-1 message in r esponse to 100 ng/mL of LPS(2). The TNF message was detectable in all groups receiving LPS(2) alone, but the highest levels of TNF mRNA were seen in LPS(1)-pretreated cells stimulated with LPS(2). Conclvsions: Endotoxin pretreatment produced increased IL-1 message that paralleled the augmentation of IL-1 protein, whereas abundant TNF message was pr esent even though TNF protein release was significantly inhibited. In this model of in vibe endotoxin tolerance, pretreatment initiates dive rgent pathways of cptokine regulation.