ABSTRACT—
Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce decreased levels of inflammatory mediators such as eicosanoids and tumor necrosis factor alpha (TNFα) in response to LPS. We hypothesized that transcriptional repression by newly synthesized proteins may be a mechanism for the reduced cellular response to a secondary challenge with LPS. THP-1 cells were desensitized after a 3.5 h or 20 h pre-exposure to LPS (1μg/mL) and subsequently challenged with LPS (10 μg/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox)-2 and TNFα mRNA was suppressed. Cycloheximide (10 μM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNFα mRNA, in LPS-tolerant cells. Transcriptional arrest with actinomycin D stabilized steady-state expression of Cox-2 mRNA in naive and tolerant cells but destabilized TNFα mRNA expression in LPS-tolerant cells. The observation that in naive cells Cox-2 and TNFα mRNA levels subside at 3 to 4 h after LPS (10 μg/mL or 1 μg/mL) suggested that LPS tolerance may occur earlier. Therefore, in subsequent experiments, the effect of LPS pretreatment for only 3.5 h was examined. This abbreviated tolerance regimen diminished secondary LPS-induced Cox-2 mRNA expression but had a lesser effect on TNFα mRNA expression. However, cycloheximide augmented both Cox-2 and TNFα mRNA expression in this group. Also, the serine/threonine phosphatase inhibitor okadaic acid augmented Cox-2 and TNFα mRNA expression in the LPS-tolerant cells. Although LPS-induced TNFα production in LPS-tolerant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNFα in tolerant and naive cells. These findings support the concept that LPS tolerance is associated with induction of proteins that alter expression of certain genes. Expression of Cox-2 mRNA appears to be particularly sensitive to down-regulation and, to a lesser extent, TNFα mRNA. However, this seems to vary depending on the LPS pretreatment regimen. The ability of a phosphatase inhibitor to induce TNFα and expression of Cox-2 and TNFα mRNA in LPS tolerance suggests that there may be alterations in phosphorylation status of signaling pathways, transcriptional mechanisms, or post-transcriptional mRNA stability.
Departments of *Physiology/Neuroscience, †Pharmacology and ‡Medicine and §Division of Clinical Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425
Address reprint requests to James A. Cook. PhD, Department of Physiology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425.
Supported in part by NIH, GM 27673, and NIH fellowship to Lawrence P. Fernando HL-07260.
Received 13 Sep 1999; first review completed 24 Oct 1999; accepted in final form 28 Jan 2000
©2000The Shock Society
